Antibodies to lymphotoxin-α

ABSTRACT

The invention provides various antibodies that bind to lymphotoxin-α, methods for making such antibodies, compositions and articles incorporating such antibodies, and their uses in treating, for example, an autoimmune disorder. The antibodies include murine, chimeric, and humanized antibodies.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.11/871,136, filed Oct. 11, 2007, now U.S. Pat. No. 7,923,011 whichclaims the benefit of priority of U.S. Provisional Application Ser. No.60/829,257 filed on 12 Oct. 2006, and of U.S. Provisional ApplicationSer. No. 60/938,999 filed on 18 May 2007, which are incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention concerns antibodies and their uses to treatautoimmune disorders. More particularly, the present invention concernsantibodies that bind to lymphotoxin-α and may also block binding of thelymphotoxin-α ligand to one or more receptors.

BACKGROUND OF THE INVENTION TNF and LT

Autoimmune diseases remain clinically important diseases in humans. Asthe name implies, autoimmune diseases wreak their havoc through thebody's own immune system. While the pathological mechanisms differ amongindividual types of autoimmune diseases, one general mechanism involvesthe binding of certain antibodies (referred to herein as self-reactiveantibodies or autoantibodies) present. Physicians and scientists haveidentified more than 70 clinically distinct autoimmune diseases,including rheumatoid arthritis (RA), multiple sclerosis (MS),vasculitis, immune-mediated diabetes, and lupus such as systemic lupuserythematosus (SLE). While many autoimmune diseases are rare—affectingfewer than 200,000 individuals—collectively, these diseases afflictmillions of Americans, an estimated five percent of the population, withwomen disproportionately affected by most diseases. The chronic natureof these diseases leads to an immense social and financial burden.

Tumor Necrosis Factor (TNF)-related proteins are recognized in the artas a large family of proteins having a variety of activities rangingfrom host defense to immune regulation to apoptosis. TNF was firstidentified as a serum-derived factor that was cytotoxic for severaltransformed cell lines in vitro and caused necrosis of certain tumors invivo. A similar factor in the superfamily was identified and referred toas lymphotoxin (“LT”). Due to observed similarities between TNF and LTin the early 1980's, it was proposed that TNF and LT be referred to asTNF-α and TNF-β, respectively. Scientific literature thus makesreference to both nomenclatures. As used in the present application, theterm “TNF” refers to TNF-α. Later research revealed two forms oflymphotoxin, referred to as LTα and LTβ. US 2005-0129614 describesanother polypeptide member of the TNF ligand super-family based onstructural and biological similarities, designated TL-5.

Members of the TNF family of proteins exist in membrane-bound forms thatact locally through cell-cell contact, or as secreted proteins. A familyof TNF-related receptors react with these proteins and trigger a varietyof signalling pathways including cell death or apoptosis, cellproliferation, tissue differentiation, and proinflammatory responses.TNF-α by itself has been implicated in inflammatory diseases, autoimmunediseases, viral, bacterial, and parasitic infections, malignancies,and/or neurodegenerative diseases and is a useful target for specificbiological therapy in diseases such as RA and Crohn's disease.

Cloning of the TNF and LTα proteins and further characterization oftheir respective biological activities reveal that the proteins differin many aspects. Aggarwal et al., Cytokines and Lipocortins inInflammation and Differentiation, Wiley-Liss, Inc. 1990, pp. 375-384.For instance, LTα is a secreted, soluble protein of approximately 20 kDa(25 kDa if N- and O-glycosylated). TNF, in contrast, has no site forglycosylation and is synthesized with an apparent transmembrane domainthat results in the original protein transcript being cell associated.Proteolysis of the cell-associated TNF protein results in the release ofthe soluble form of the protein having a molecular weight ofapproximately 19 kDa. TNF is produced primarily by activatedmacrophages, whereas LT is produced by activated lymphocytes. Wong etal., Tumor Necrosis Factors: The Molecules and their Emerging Role inMedicine, Beutler, B., ed., Raven Press (1991), pp. 473-484. Thesequences encoding TNF and LTα also differ. TNF and LTα share onlyapproximately 32% amino acid sequence identity. Regarding the differentbiological activities of TNF and LTα, TNF increases production ofendothelial-cell interleukin-1 (“IL-1”), whereas LTα has little effectthereon. Further, TNF induces production ofmacrophage-colony-stimulating factor from macrophages, whereas LTα hasno effect thereon. These and other biological activities are discussedin Aggarwal, Tumor Necrosis Factors: Structure, Function and Mechanismof Action, Aggarwal and Vicek, eds. (1992), pp. 61-78.

TNF and LTα are described further in the review articles by Spriggs,“Tumor Necrosis Factor Basic Principles and Preclinical Studies,”Biologic Therapy of Cancer, DeVita et al., eds., J.B. Lippincott Company(1991) Ch. 16, pp. 354-377; Ruddle, Current Opinion in Immunology,4:327-332 (1992); Wong et al., “Tumor Necrosis Factor,” Human Monocytes,Academic Press (1989), pp. 195-215; and Paul et al., Ann. Rev. Immunol.,6:407-438 (1988).

In non-tumor cells, TNF and TNF-related cytokines are active in avariety of immune responses. Both TNF and LTα ligands bind to andactivate TNF receptors (p55 or p60 and p75 or p80; herein called“TNF-R”).

Cell-surface LT complexes have been characterized in CD4+ T cellhybridoma cells (II-23.D7), which express high levels of LT (Browning etal., J. Immunol., 147: 1230-1237 (1991); Androlewicz et al., J. Biol.Chem., 267: 2542-2547 (1992)). The expression and biological roles ofLTβ-R, LT subunits, and surface LT complexes are reviewed in Ware etal., “The ligands and receptors of the lymphotoxin system”, in Pathwaysfor Cytolysis, Current Topics Microbiol. Immunol., Springer-Verlag, pp.175-218 (1995).

LTα expression is induced and LTα secreted primarily by activated T andB lymphocytes and natural killer (NK) cells. Among the T helper cellsubclasses, LTα appears to be produced by Th1 but not Th2 cells. LTα hasalso been detected in melanocytes. LTβ (also called p33) has beenidentified on the surface of T lymphocytes, T cell lines, B cell linesand lymphokine-activated killer (LAK) cells. Studies have shown that LTDis not functional in the absence of LTα.

LTα exists either as a homotrimer (LTα3) or a heterotrimer with LTβ.These heterotrimers contain either two subunits of LTα and one subunitof LTβ (LTα2β1), or one subunit of LTα and two of LTβ (LTα1β2).

The only known cell-surface receptors for the LTα homotrimer are the twoTNF receptors, p55 and p75. However, the LTαβ heterotrimer, LTα1β2, doesnot bind to the TNF receptors and instead binds to a member of the TNFreceptor superfamily, lymphotoxin β receptor (referred to herein asLTβ-R). The heterotrimeric form LTα2β1 likely binds TNF receptors.

LTβ-R has a well-described role both in the development of the immunesystem and in the functional maintenance of a number of cells in theimmune system, including follicular dendritic cells and a number ofstromal cell types (Matsumoto et al., Immunol. Rev. 156:137 (1997)).Known ligands to the LTβ-R include not only LTα1β2, but also a secondligand called LIGHT (Mauri et al., Immunity 8:21 (1998)). Activation ofLTβ-R has been shown to induce the apoptotic death of certain cancercell lines in vivo (U.S. Pat. No. 6,312,691). Humanized antibodies toLTβ-R and methods of use thereof are provided in US 2004-0058394 andstated as being useful for treating or reducing the advancement,severity, or effects of neoplasia in humans. Further, EP 1585547 (WO2004/058183) (LePage and Gill) discloses combination therapies thatinclude a composition that activates LTβ-R signaling in combination withone or more other chemotherapeutic agents, as well as therapeuticmethods and screening methods for identifying agents that in combinationwith a LTβ-R agonist agent have an additive effect on tumor inhibition.

LT is important for lymphoneogenesis, as evident from knockout mice. SeeFutterer et al. Immunity, 9 (1): 59-70 (1998), showing that micedeficient in LTβ-R lacked lymph nodes and Peyer's patches and alsoshowing impaired antibody affinity maturation. Rennert et al., Immunity,9 (1): 71-9 (1998) reported that an agonist monoclonal antibody againstLTβ-R restored the ability to make lymph nodes in LTα knockout mice. Seealso Wu et al., J. Immunology, 166 (3): 1684-9 (2001) and Endres et al.,J. Exp. Med., 189 (1): 159-68 (1999); Dohi et al., J. Immunology, 167(5): 2781-90 (2001); and Matsumoto et al., J. Immunology, 163 (3):1584-91 (1999). Korner et al. Eur. J. Immun., 27 (10): 2600-9 (1997)reported that mice lacking both TNF and LT showed retarded B-cellmaturation and serum immunoglobulin deficiencies, whereas mice lackingonly TNF showed no such deficiencies.

In addition, LT is important for inflammation. LTα is overexpressed inthe pancreas of RIP.LTα transgenic mice, which have shown inflammation,increased chemokine expression, and a lymphoid-like structure, and inwhich overexpression of LTβ alone has demonstrated no additionalinflammation. Further, LTα-deficient mice exhibit impaired TNF-αproduction, and defective splenic architecture and function are restoredwhen such mice are crossed to TNF-transgene (Kollias, J. Exp. Med.,188:745 (1998); Chaplin, Ann Rev Imm 17:399 (1999)), and decreased TNFlevels are restored after pathogenic challenge (Eugster, Eur. J. Immun.31:1935 (2001)).

When TNF-α or LTα₃ interacts with the TNF receptors TNFRI and/or TNFRII,the result is proinflammatory responses and/or apoptosis. When LTα1β2interacts with the receptor LTβ-R, the result is lymphoneogenesis andinduction of chemokines and adhesion molecules. Autoimmune diseases areassociated with lymphoneogenesis and inflammatory responses, and thereis increased LT expression in patients with autoimmune disease,including MS, inflammatory bowel disease (IBD), and RA (Weyand et al.,Curr. Opin. Rheumatol., 15: 259-266 (2003); Selmaj et al., J. Clin.Invest., 87: 949-954 (1991); Matusevicius et al., J. Neuroimm., 66:115-123 (1996); Powell et al., International Immunology, 2 (6): 539-44(1990); Zipp et al., Annals of Neurology, 38/5: 723-730 (1995); Voskuhlet al., Autoimmunity 15 (2): 137-43 (1993); Selmaj et al., J.Immunology, 147: 1522-29 (1991); Agyekum et al., Journal Pathology, 199(1): 115-21 (2003); and Takemura et al., J. Immunol., 167: 1072 (2001)).

As to MS specifically, serum LTα correlates with lesions/disease burdenin MS (Kraus et al., Acta Neurologica Scandinavica, 105 (4): 300-8(2002)). LTα is involved in demyelination but not remyelination in an invivo cuprizone model, whereas TNF-α is required for both (Plant et al.,Glia 49:1-14 (2005)).

As to RA specifically, levels of human LTα3 and TNF-α in RA patients areelevated over those of normal donors (Stepien, Eur Cytokine Net 9: 145(1998)). The roles of LTα in RA include: serum LTα is present in some RApatients, increased LTα protein is present in synovium, the LT pathwayis associated with ectopic lymphoneogenesis in synovium, and there isincreased LTβ-R expression on fibroblast-like synoviocytes in RApatients. In addition, a case report discloses that neutralizing LTα3 isbeneficial for an infliximab-resistant RA patient (Buch et al., Ann.Rheum. Dis., 63: 1344-46 (2004)). Also, Han et al., Arthrit. Rheumat.,52: 3202-3209 (2005) describes that blockading the LT pathwayexacerbates autoimmune arthritis by enhancing the Th1 response.

Preclinical efficacy for prevention and treatment with LTβR-Ig incollagen-induced arthritis (CIA) is shown in Fava et al., J. Immunology,171 (1): 115-26 (2003). Further, LTα-deficient mice are resistant toexperimental autoimmune encephalomyelitis (EAE) (Suen et al., J. Exp.Med, 186: 1233-40 (1997); Sean Riminton et al., J. Exp. Med, 187 (9):1517-28 (1998)). There is also published efficacy of LTβR-Ig in EAE(Gommerman et al., J. Clin. Invest, 112 (5): 755-67 (2003)). Also,LTβR-Ig disrupts lymphogenesis in mice. Mackay et al., Europ. J.Immunol. 27 (8): 2033-42 (1997)). Further, administration of LTβR-Igdecreases insulin-dependent diabetes mellitus (IDDM) in non-obesediabetic mice (Wu et al., J. Exp. Med, 193 (11): 1327-32 (2001)). Therole of LT in lymphogenesis in non-human primates was investigated byGommerman et al., J. Clin. Invest. 110 (9): 1359-69 (2002) usingLTβR-Ig. Further, LTα-deficient mice are less susceptible to M. bovisBCG than TNF-α-deficient mice. Eugster et al., Europ. J. Immunol., 31:1935 (2001).

LT has also been used to treat cancer. See U.S. Pat. No. 5,747,023.

Antibodies

Antibodies are proteins that exhibit binding specificity to a specificantigen. Native antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light-chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) both in the light-chain and the heavy-chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FRs). The variable domains of native heavyand light chains each comprise four FRs, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FRs and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. Theheavy-chain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. Of the humanimmunoglobulin classes, only human IgG1, IgG2, IgG3, and IgM are knownto activate complement, and human IgG1 and IgG3 mediateantibody-dependent cell-mediated cytotoxicity (ADCC) more effectivelythan IgG2 and IgG4.

Two main approaches have been used to develop therapeutic compounds thatinhibit TNF-α. The first, exemplified by infliximab, a chimericmonoclonal antibody to TNF-α also known as REMICADE®, is to neutralizeTNF-α action by using a specific monoclonal antibody of high affinityand potency to prevent binding of TNF-α to its receptors. The second,exemplified by etanercept, also known as ENBREL®, is to inhibit TNF-α byusing a TNF receptor-based molecule that functions as a “decoy” toreduce the binding of TNF-α to its natural receptors. Although bothtypes of molecules can prevent the binding of TNFα to its receptors,receptor-based inhibitors such as etanercept will also prevent receptorbinding of LTα.

In the patent literature, antibodies to cachectin (TNF), disclosed by EP212489, are reported as useful in diagnostic immunoassays and in therapyof shock in bacterial infections. EP 218868 discloses monoclonalantibodies to human TNF and their uses. EP 288,088 discloses anti-TNFantibodies, and their utility in immunoassay diagnosis of pathologies,in particular Kawasaki's pathology and bacterial infection. Anti-TNFantibodies are also described, for example, in EP 1585477 (Giles-Komaret al.); US 2006-0147452; US 2006-0222646; and US 2006-0121037. A methodof treating RA by using a single-domain antibody polypeptide constructthat antagonizes human TNFα's binding to a receptor is described in US2005-0271663. Methods of treating RA using anti-TNF receptor fusionproteins are described in US 2005-0260201. Methods for treating aTNF-mediated disease using a composition comprising methotrexate and ananti-TNF antibody, including RA, Crohn's disease, and acute and chronicimmune diseases associated with transplantation, are described in EP1593393. Antibodies that bind to TNF-R include those described in U.S.Pat. No. 7,057,022. Novel proteins with TNF-alpha antagonist activityare described in U.S. Pat. No. 7,056,695. US 2006-0147448 disclosestreatment of immunological renal disorders by LT pathway inhibitors.

LTβ and LTβ/LTα complexes and LTβ-R and antibodies thereto as well asLTβ blocking agents are described, for example, in WO 1992/00329; WO1994/13808; U.S. Pat. Nos. 5,661,004; 5,795,964; EP 0954333; U.S. Pat.Nos. 5,670,149; 5,925,351; US 2005-0037003; U.S. Pat. Nos. 6,403,087;6,669,941; 6,312,691; 7,001,598; 7,030,080; US 2006-0222644; and US2005-0163747. The application US 2006-0134102 discloses LTβ-R agents incombination with chemotherapeutic agents. Antibodies to the LTβ-R aredescribed in US 2005-0281811. See also WO 2006/114284 regarding use ofLTβ-R antibodies to prevent and/or treat obesity and obesity-relateddisorders. Humanized antibodies specific for the LTβ-R alone or incombination with chemotherapeutic agent(s) in therapeutic methods aredisclosed in EP 1539793. LTα and antibodies thereto are described, forexample, in U.S. Pat. Nos. 4,959,457; 5,824,509; 4,920,196; and5,683,688; US 2005-0266004 (WO 2005/067477) and U.S. Pat. No. 5,188,969(EP 347728B1). See also US 2002-0039580; WO 2004/039329; and US2002-0197254 regarding LTβ or LTβ-R technology. Multivalent antibodies(that bind the TNF receptor superfamily) are described, for example, inUS 2002-0004587 and US 2006-0025576.

There is a continuing need in the art to produce antibodies, inparticular, therapeutic antibodies having improved function, such asanti-LTα antibodies or fragments thereof that block LTβ, since LTα hasmore interactions with the various receptors than TNF-α or LTβ alone.

SUMMARY OF THE INVENTION

The invention is in part based on the identification of a variety ofantagonists of the LT biological pathway, which is a biological/cellularprocess presenting as an important therapeutic target. The inventionprovides compositions and methods based on interfering with LTαactivation, including but not limited to interfering with LTα binding tovarious receptors.

Accordingly, the invention is as claimed. In one aspect, the inventionprovides an isolated anti-lymphotoxin-α (LTα) antibody comprising atleast one complementarity-determining region (CDR) sequence selectedfrom the group consisting of:

(a) a CDR-L1 sequence comprising amino acids A1-A11, wherein A1-A11 isKASQAVSSAVA (SEQ ID NO:1) or RASQAVSSAVA (SEQ ID NO:2), or comprisingamino acids A1-A17, wherein A1-A17 is KSSQSLLYSTNQKNFLA (SEQ ID NO:3) orKSSQSLLYSANQKNFLA (SEQ ID NO:4) or KSSQSLLYSTNQKNALA (SEQ ID NO:6),where N is any amino acid (chimeric 2C8 or humanized 2C8.v2/2C8.vX orchimeric 3F12/humanized 3F12.v5/humanized 3F12.v14, or 3F12 clone 14 or17, respectively);

(b) a CDR-L2 sequence comprising amino acids B1-B7, wherein B1-B7 isSASHRYT (SEQ ID NO:7) or WASTRDS (SEQ ID NO:8) (chimeric 2C8/humanized2C8.v2/humanized 2C8.vX or chimeric 3F12/humanized 3F12.v5/humanized3F12.v14, respectively);

(c) a CDR-L3 sequence comprising amino acids C1-C9, wherein C1-C9 isQQHYSTPWT (SEQ ID NO:9) or QENYSTPWT (SEQ ID NO:11) or QQYYSYPRT (SEQ IDNO:13) or QQYASYPRT (SEQ ID NO:14) or QQYYAYPRT (SEQ ID NO:15), where Nis any amino acid (chimeric 2C8/humanized 2C8.v2 or 2C8 clone G7 orchimeric 3F12/humanized 3F12.v5/humanized 3F12.v14 or 3F12 clone 20 or3F12 clone 21, respectively);

(d) a CDR-H1 sequence comprising amino acids D1-D10, wherein D1-D10 isGYTFTSYVIH (SEQ ID NO:16) or GYTFSSYWIE (SEQ ID NO:17) (chimeric2C8/humanized 2C8.v2/humanized 2C8.vX or chimeric 3F12/humanized3F12.v5/humanized 3F12.v14, respectively);

(e) a CDR-H2 sequence comprising amino acids E1-E17, wherein E1-E17 isYNNPYNDGTNYNEKFKG (SEQ ID NO:1-8) or EISPGSGSTNYNEEFKG (SEQ ID NO:19) orYNNPYNAGTNYNEKFKG (SEQ ID NO:101) or EINPGSGSTIYNEKFKG (SEQ ID NO:110),wherein N is any amino acid (chimeric 2C8/humanized 2C8.v2 or chimeric3F12/humanized 3F12.v5, or humanized 2C8.vX, or humanized 3F12.v14,respectively); and

(f) a CDR-H3 sequence comprising amino acids F1-F9, wherein F1-F9 isPTMLPWFAY (SEQ ID NO:20), or comprising amino acids F1-F5, wherein F1-F5is GYHGY (SEQ ID NO:21) or GYHGA (SEQ ID NO:22) (chimeric 2C8/humanized2C8.v2/humanized 2C8.vX or chimeric 3F12/humanized 3F12.v5/humanized3F12.v14 or 3F12 clone 12, respectively).

Preferably, SEQ ID NO:3 is KSSQSLLYSTAQKNFLA (SEQ ID NO:5) (3F12 clone15). In another preferred aspect, SEQ ID NO:11 is QESYSTPWT (SEQ IDNO:10) (2C8 clone A8/humanized 2C8.vX) or QEVYSTPWT (SEQ ID NO:12) (2C8clone H6).

In a further preferred embodiment, the CDR-L1 sequence is SEQ ID NO:2 or3 or 4 or 6.

In another preferred aspect, the antibody comprises either (i) all ofthe CDR-L1 to CDR-L3 amino acid sequences of SEQ ID NOS:1 or 2 and 7 and9, or of SEQ ID NOS:1 or 2 and 7 or 8 and 11, or of SEQ ID NOS:3, 8, and13, or of SEQ ID NOS:4, 5, or 6, 8, and 13, or of SEQ ID NOS:3, 8, and14 or 15, or of SEQ ID NOS:4, 5, or 6, 8, and 14 or 15; or (ii) all ofthe CDR-H1 to CDR-H3 amino acid sequences of SEQ ID NOS:16, 18, and 20,or all of SEQ ID NOS:16, 101, and 20, or all of SEQ ID NOS:17, 19, and21 or 22, or all of SEQ ID NOS:17, 110, and 21.

In a still further preferred aspect, the antibody comprises either allof SEQ ID NOS:1 or 2 and 7 and 9, or all of SEQ ID NOS:16, 18, and 20,or all of SEQ ID NOS:16, 101, and 20. In an alternative embodiment, theantibody comprises either all of SEQ ID NOS:3, 8, and 13, or all of SEQID NOS:17, 19, and 21 or 22. In an alternative embodiment, the antibodycomprises either all of SEQ ID NOS:4, 8, and 14, or all of SEQ IDNOS:17, 110, and 21. In other embodiments, the antibody comprises (i)all of the CDR-L1 to CDR-L3 amino acid sequences of SEQ ID NOS:1 or 2, 7and 9, or of SEQ ID NOS:1 or 2 and 7 or 8 and 11, or of SEQ ID NOS:3, 8,and 13, or of SEQ ID NOS:4, 5, or 6, 8, and 13, or of SEQ ID NOS:3, 8,and 14 or 15, or of SEQ ID NOS:4, 5, or 6, 8, and 14 or 15; and (ii) allof the CDR-H1 to CDR-H3 amino acid sequences of SEQ ID NOS:16, 18, and20, or of SEQ ID NOS:16, 101, and 20, or of SEQ ID NOS:17, 19, and 21 or22, or of SEQ ID NOS:17, 110, and 21.

In addition, the antibodies herein are preferably chimeric or humanized,most preferably humanized. A humanized antibody of the invention maycomprise one or more suitable human and/or human consensus non-CDR(e.g., framework) sequences in its heavy- and/or light-chain variabledomains, provided the antibody exhibits the desired biologicalcharacteristics (e.g., a desired binding affinity). Preferably, at leasta portion of such humanized antibody framework sequence is a humanconsensus framework sequence.

In some embodiments, one or more additional modifications are presentwithin the human and/or human consensus non-CDR sequences. In oneembodiment, the heavy-chain variable domain of an antibody of theinvention comprises at least a portion of (preferably all of) a humanconsensus framework sequence, which in one embodiment is the subgroupIII consensus framework sequence. In one embodiment, an antibody of theinvention comprises a variant subgroup III consensus framework sequencemodified at least one amino acid position. For example, in oneembodiment, a variant subgroup III consensus framework sequence maycomprise a substitution at one or more of positions 71, 73, and/or 78.In one embodiment, said substitution is R71A, N73T, and/or N78A, in anycombination thereof, preferably all three. In another embodiment, theseantibodies comprise or further comprise at least a portion of(preferably all of) a human κ light-chain consensus framework sequence.In a preferred embodiment, an antibody of the invention comprises atleast a portion of (preferably all of) a human κ subgroup I frameworkconsensus sequence.

The amino acid position/boundary delineating a CDR of an antibody canvary, depending on the context and the various definitions known in theart (as described below). Some positions within a variable domain may beviewed as hybrid hypervariable positions in that these positions can bedeemed to be within a CDR under one set of criteria while being deemedto be outside a CDR under a different set of criteria. One or more ofthese positions can also be found in extended CDRs (as further definedbelow). The invention provides antibodies comprising modifications inthese hybrid hypervariable positions. In one embodiment, these hybridhypervariable positions include one or more of positions 26-30, 33-35B,47-49, 57-65, 93, 94, and 102 in a heavy-chain variable domain. In oneembodiment, these hybrid hypervariable positions include one or more ofpositions 24-29, 35-36, 46-49, 56, and 97 in a light-chain variabledomain. In one embodiment, an antibody of the invention comprises avariant human subgroup consensus framework sequence modified at one ormore hybrid hypervariable positions.

In another preferred aspect, the antibody binds to LTα3 and blocks theinteraction of LTα3 with tumor necrosis factor receptor-1 (TNFRI) andtumor necrosis factor receptor-2 (TNFRII). Preferably, it binds also toone or more LTαβ complexes and especially on the cell surface.Preferably, it also blocks the function of one or more LTαβ complexes.Also, it preferably decreases levels of inflammatory cytokinesassociated with rheumatoid arthritis in an in vitro arthritis assay suchas an in vitro collagen-induced arthritis assay or an in vitroantibody-induced arthritis assay.

In some aspects, such as the murine S5H3 antibody, the antibody does notblock the interaction of LTαβ with LTβ-R. In other aspects, the antibodydoes block the interaction of LTαβ with LTβ-R. Preferably, the antibodymodulates LTαβ-expressing cells. In other embodiments, the anti-LTαantibody substantially neutralizes at least one activity of at least oneLTα protein. Preferably, the antibody herein targets any cell expressingLTβ, and more preferably depletes LTβ-positive or -secreting cells.

The preferred antibody binds LTα with an affinity of at least about10⁻¹²M (picomolar levels), and more preferably at least about 10⁻¹³ M.Also preferred is an IgG antibody, more preferably human IgG. Human IgGencompasses any of the human IgG isotypes of IgG1, IgG2, IgG3, and IgG4.Murine IgG encompasses the isotypes of IgG1, 2a, 2b, and 3. Morepreferably, the murine IgG is IgG2a and the human IgG is IgG1. In otherpreferred embodiments of the human IgG, the VH and VL sequences providedare joined to human IgG1 constant region.

In another embodiment, the invention provides an anti-LTα antibodyhaving a light-chain variable domain comprising SEQ ID NO:23 or 24, or aheavy-chain variable domain comprising SEQ ID NO:25 or 26, or havinglight-chain and heavy-chain variable domains comprising both SEQ IDNOS:23 and 25, or comprising both SEQ ID NOS:24 and 26.

In a still further embodiment, the invention provides an anti-LTαantibody having a light-chain variable domain comprising SEQ ID NO:27 or28, or a heavy-chain variable domain comprising SEQ ID NO:29 or 30 or31, or having light-chain and heavy-chain variable domains comprisingboth SEQ ID NOS:27 and 29, or comprising both SEQ ID NOS:27 and 30, orcomprising both SEQ ID NOS:27 and 31, or comprising both SEQ ID NOS:28and 30, or comprising both SEQ ID NOS:28 and 31.

In a still further aspect, the invention provides an anti-LTα antibodyhaving a light-chain variable domain comprising SEQ ID NO:102, or aheavy-chain variable domain comprising SEQ ID NO:103, or havinglight-chain and heavy-chain variable domains comprising both SEQ IDNOS:102 and 103.

In a still further aspect, the invention provides an anti-LTα antibodyhaving a light-chain variable domain comprising SEQ ID NO:108, or aheavy-chain variable domain comprising SEQ ID NO:109, or havinglight-chain and heavy-chain variable domains comprising both SEQ IDNOS:108 and 109.

In other preferred embodiments, the antibody has an Fc region. In oneaspect, such Fc region is a wild-type (or native-sequence) Fc region. Inanother embodiment, the antibody further comprises one or more aminoacid substitutions in its Fc region that result in the polypeptideexhibiting at least one of the following properties: increased FcγRbinding, increased antibody-dependent cell-mediated cytotoxicity (ADCC),increased complement-dependent cytotoxicity (CDC), decreased CDC,increased ADCC and CDC function, increased ADCC but decreased CDCfunction, increased FcRn binding, and increased serum half life, ascompared to an antibody having a native-sequence Fc region. Morepreferably, the antibody further comprises one or more amino acidsubstitutions in its Fc region that result in it having increased ADCCfunction as compared to the same antibody having a native-sequence Fcregion.

In a particularly preferred embodiment, the antibody has amino acidsubstitutions in its Fc region at any one or any combination ofpositions that are 268D, or 298A, or 326D, or 333A, or 334A, or 298Atogether with 333A, or 298A together with 334A, or 239D together with332E, or 239D together with 298A and 332E, or 239D together with 268Dand 298A and 332E, or 239D together with 268D and 298A and 326A and332A, or 239D together with 268D and 298A and 326A and 332E, or 239Dtogether with 268D and 283L and 298A and 332E, or 239D together with268D and 283L and 298A and 326A and 332E, or 239D together with 330L and332E and 272Y and 254T and 256E, or 250Q together with 428L, or 265A, or297A, wherein the 265A substitution is in the absence of 297A and the297A substitution is in the absence of 265A. In one particularembodiment, the Fc region has from one to three such amino acidsubstitutions, for example, substitutions at positions 298, 333, and334, and more preferably the combination of 298A, 333A, and 334A. Theletter after the number in each of these designations represents thechanged amino acid at that position.

Such anti-LTα antibodies effect varying degrees of disruption of theLTα/LTβ signaling pathway. For example, in one embodiment, the inventionprovides an anti-LTα antibody (preferably humanized) wherein themonovalent affinity of the antibody to human LTα (e.g., affinity of theantibody as a Fab fragment to human LTα) is about the same as or greaterthan that of a murine antibody (e.g., affinity of the murine antibody asa Fab fragment to human LTα) produced by a hybridoma cell line depositedunder American Type Culture Collection Accession Number (ATCC) PTA-7538(hybridoma murine Lymphotoxin alpha2 beta1 s5H3.2.2). The monovalentaffinity is preferably expressed as a Kd value and/or is measured byoptical biosensor that uses surface plasmon resonance (SPR) (BIACORE®technology) or radioimmunoassay.

Further antibodies herein include those with any of the properties abovehaving reduced fusose relative to the amount of fucose on the sameantibody produced in a wild-type Chinese hamster ovary cell. Morepreferred are those antibodies having no fucose.

In another embodiment, the invention provides an antibody compositioncomprising the antibodies described herein having an Fc region, whereinabout 20-100% of the antibodies in the composition comprise a maturecore carbohydrate structure in the Fc region that lacks a fucose.Preferably, such composition comprises antibodies having an Fc regionthat has been altered to change one or more of the ADCC, CDC, orpharmacokinetic properties of the antibody compared to a wild-type IgGFc sequence by substituting an amino acid selected from the groupconsisting of A, D, E, L, Q, T, and Y at any one or any combination ofpositions of the Fc region selected from the group consisting of: 238,239, 246, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269,270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295,296, 297, 298, 301, 303, 305, 307, 309, 312, 314, 315, 320, 322, 324,326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373,376, 378, 382, 388, 389, 398, 414, 416, 419, 428, 430, 434, 435, 437,438 and 439.

The above-described antibody composition is more preferably one whereinthe antibody further comprises an Fc substitution that is 268D or 326Dor 333A together with 334A, or 298A together with 333A, or 298A togetherwith 334A, or 239D together with 332E, or 239D together with 298A and332E, or 239D together with 268D and 298A and 332E, or 239D togetherwith 268D and 298A and 326A and 332A, or 239D together with 268D and298A and 326A and 332E, or 239D together with 268D and 283L and 298A and332E, or 239D together with 268D and 283L and 298A and 326A and 332E, or239D together with 330L and 332E, wherein the letter after the number ineach of these designations represents the changed amino acid at thatposition.

The above-described antibody composition is additionally preferably onewherein the antibody binds an FcγRIII. The composition is preferably onewherein the antibody has ADCC activity in the presence of human effectorcells or has increased ADCC activity in the presence of human effectorcells compared to the otherwise same antibody comprising a humanwild-type IgG1Fc. The composition is also preferably one wherein theantibody binds the FcγRIII with better affinity, or mediates ADCC moreeffectively, than a glycoprotein with a mature core carbohydratestructure including fucose attached to the Fc region of theglycoprotein. In addition, the composition is preferably one wherein theantibody has been produced by a Chinese hamster ovary (CHO) cell,preferably a Lec13 cell. The composition is also preferably one whereinthe antibody has been produced by a mammalian cell lacking afucosyltransferase gene, more preferably the FUT8 gene.

In one embodiment, the above-described composition is one wherein theantibody is free of bisecting N-acetylglucosamine (GlcNAc) attached tothe mature core carbohydrate structure. In an alternative embodiment,the composition is one wherein the antibody has bisecting GlcNAcattached to the mature core carbohydrate structure.

In another aspect, the above-described composition is one wherein theantibody has one or more galactose residues attached to the mature corecarbohydrate structure. In an alternative embodiment, the composition isone wherein the antibody is free of one or more galactose residuesattached to the mature core carbohydrate structure.

In a further aspect, the above-described composition is one wherein theantibody has one or more sialic acid residues attached to the maturecore carbohydrate structure. In an alternative aspect, the compositionis one wherein the antibody is free of one or more sialic acid residuesattached to the mature core carbohydrate structure.

The above-described composition preferably comprises at least about 2%afucosylated antibodies, more preferably at least about 4% afucosylatedantibodies, still more preferably at least about 10% afucosylatedantibodies, even more preferably at least about 19% afucosylatedantibodies, and most preferably about 100% afucosylated antibodies.

Also included herein is an anti-idiotype antibody that specificallybinds any of the antibodies herein.

A therapeutic agent for use in a host subject preferably elicits littleto no immunogenic response against the agent in said subject. In oneembodiment, the invention provides a chimeric or humanized antibody thatelicits and/or is expected to elicit a human anti-mouse antibodyresponse (HAMA) at a substantially reduced level compared to a murineantibody in a host subject. In another example, the invention provides achimeric or humanized antibody that elicits and/or is expected to elicitminimal or no human anti-mouse antibody response (HAMA). In one example,an antibody of the invention elicits an anti-mouse antibody responsethat is at or below a clinically acceptable maximum level.

Antibodies of the invention can be used to modulate one or more aspectsof LTα-associated effects, including but not limited to LTα receptoractivation, downstream molecular signaling, cell proliferation, cellmigration, cell survival, cell morphogenesis, and angiogenesis. Theseeffects can be modulated by any biologically relevant mechanism,including disruption of ligand (e.g., LTα), binding to and blocking theLTα3 receptor or one or both of the heterodimeric receptors, as well asreceptor phosphorylation, and/or receptor multimerization.

In another aspect, the invention provides a method of inhibitingLTα-activated cell proliferation, said method comprising contacting acell or tissue with an effective amount of one or more of the antibodiesof the invention.

In a preferred embodiment, such LTα-actived cell proliferation is due toan autoimmune disorder, more preferably RA, MS, Sjogren's syndrome,lupus, myasthenia gravis, or IBD, or cancer, especially breast cancer,lung cancer, prostate cancer, a lymphoma, or leukemia. In anotherpreferred embodiment, the method further comprises contacting the cellor tissue with a second medicament, wherein the first medicament is oneor more of the antibodies herein. In a still further preferredembodiment, the cell or tissue is a mammalian cell or tissue, morepreferably human, and still more preferably the method is an in vivomethod. In such preferred in vivo method, preferably, the subject havingthe cell or tissue, most preferably a human subject, is administered theeffective amount of the antibodies herein, such as by intravenous orsubcutaneous administration.

In another embodiment, the invention provides a method of treating anautoimmune disorder in a subject comprising administering to the subjectan effective amount of the antibody of the invention. Preferably, theautoimmune disorder is selected from the group consisting of rheumatoidarthritis (RA), lupus, Wegener's disease, inflammatory bowel disease(IBD), idiopathic thrombocytopenic purpura (ITP), thromboticthrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiplesclerosis (MS), psoriasis, IgA nephropathy, IgM polyneuropathies,myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome,Sjögren's syndrome, glomerulonephritis, Hashimoto's thyroiditis, Graves'disease, helicobacter-pylori gastritis, and chronic hepatitis C.

Preferably, the autoimmune disorder is RA, MS, Sjögren's syndrome,lupus, myasthenia gravis, or IBD, more preferably the autoimmunedisorder is RA, IBD, lupus, or MS. More preferably, the lupus issystemic lupus erythematosus or lupus nephritis, and the IBD is Crohn'sdisease or ulcerative colitis.

In other aspects, the antibody is a naked antibody. In a further aspect,the antibody is conjugated with another molecule, such as a cytotoxicagent.

In another aspect, the method is such that the antibody is administeredintravenously. In an alternative aspect, the antibody is administeredsubcutaneously.

In another embodiment, the subject has RA and the antibody induces amajor clinical response in the subject.

In a further aspect, the subject has an abnormal level of one or moreregulatory cytokines, anti-nuclear antibodies (ANA), anti-rheumatoidfactor (RF) antibodies, creatinine, blood urea nitrogen,anti-endothelial antibodies, anti-neutrophil cytoplasmic antibodies(ANCA), infiltrating CD20 cells, anti-double stranded DNA (dsDNA)antibodies, anti-Sm antibodies, anti-nuclear ribonucleoproteinantibodies, anti-phospholipid antibodies, anti-ribosomal P antibodies,anti-Ro/SS-A antibodies, anti-Ro antibodies, anti-La antibodies,antibodies directed against Sjögren's-associated antigen A or B (SS-A orSS-B), antibodies directed against centromere protein B (CENP B) orcentromere protein C (CENP C), autoantibodies to ICA69, anti-Smithantigen (Sm) antibodies, anti-nuclear ribonucleoprotein antibodies,anti-ribosomal P antibodies, autoantibodies staining the nuclear orperinuclear zone of neutrophils (pANCA), anti-Saccharomyces cerevisiaeantibodies, cross-reactive antibodies to GM1 ganglioside or GQ1bganglioside, anti-acetylcholine receptor (AchR), anti-AchR subtype, oranti-muscle specific tyrosine kinase (MuSK) antibodies, serumanti-endothelial cell antibodies, IgG or anti-desmoglein (Dsg)antibodies, anti-centromere, anti-topoisomerase-1 (Scl-70), anti-RNApolymerase or anti-U3-ribonucleoprotein (U3-RNP) antibodies,anti-glomerular basement membrane (GBM) antibodies, anti-glomerularbasement membrane (GBM) antibodies, anti-mitochondrial (AMA) oranti-mitochondrial M2 antibodies, anti-thyroid peroxidase (TPO),anti-thyroglobin (TG) or anti-thyroid stimulating hormone receptor(TSHR) antibodies, anti-nucleic (AN), anti-actin (AA) or anti-smoothmuscle antigen (ASM) antibodies, IgA anti-endomysial, IgA anti-tissuetransglutaminase, IgA anti-gliadin or IgG anti-gliadin antibodies,anti-CYP21A2, anti-CYP11A1 or anti-CYP17 antibodies,anti-ribonucleoprotein (RNP), or myosytis-specific antibodies,anti-myelin associated glycoprotein (MAG) antibodies, anti-hepatitis Cvirus (HCV) antibodies, anti-GM1 ganglioside, anti-sulfate-3-glycuronylparagloboside (SGPG), or IgM anti-glycoconjugate antibodies, IgManti-ganglioside antibody, anti-thyroid peroxidase (TPO),anti-thyroglobin (TG) or anti-thyroid stimulating hormone receptor(TSHR) antibodies, anti-myelin basic protein or anti-myelinoligodendrocytic glycoprotein antibodies, IgM rheumatoid factorantibodies directed against the Fc portion of IgG, anti-Factor VIIIantibodies, or a combination thereof.

In another preferred aspect of this method, the antibody is administeredno more than about once every other week, more preferably about once amonth, to the subject.

In another embodiment, a second medicament, wherein the antibody is afirst medicament, is administered to the subject in an effective amountto treat the subject in the methods above. Such second medicament ispreferably an immunosuppressive agent, an antagonist that binds a B-cellsurface marker, a BAFF antagonist, a disease-modifying anti-rheumaticdrug (DMARD), an integrin antagonist, a non-steroidal anti-inflammatorydrug (NSAID), a cytokine antagonist, or a combination thereof.Additionally, it may be a hyaluronidase glycoprotein as an activedelivery vehicle. Most preferably it is a DMARD or methotrexate.

In another embodiment, the subject has never been previously treatedwith a medicament for the disorder. In a further aspect, the subject hasnever been previously treated with a TNF antagonist.

In an alternative embodiment, the subject has been previously treatedwith a medicament for the disorder, preferably with a TNF antagonist(such as an anti-TNF antibody or a TNF receptor-Ig such as etanercept)or a DMARD.

In another embodiment, the invention provides a method of treating RA ina subject comprising administering to the subject an effective amount ofan antibody of this invention. In this method, preferably the antibodyinduces a major clinical response in the subject. More preferably, thesubject has been treated with a medicament for the disorder, preferablywith a TNF antagonist (such as an anti-TNF antibody or a TNF receptor-Igsuch as etanercept) or a DMARD. In another preferred aspect of thismethod, the antibody is administered no more than about once every otherweek, more preferably about once a month, to the subject.

In another embodiment of this RA treatment method, a second medicament,wherein the antibody is a first medicament, is administered to thesubject being treated for RA in an effective amount to treat thesubject. Such second medicament is preferably an immunosuppressiveagent, an antagonist that binds a B-cell surface marker, a BAFFantagonist, a disease-modifying anti-rheumatic drug (DMARD), an integrinantagonist, a non-steroidal anti-inflammatory drug (NSAID), a cytokineantagonist, or a combination thereof. Additionally, it may be ahyaluronidase glycoprotein as an active delivery vehicle. Mostpreferably it is a DMARD or methotrexate. Preferably, the antibody usedfor treating RA is administered subcutaneously or intravenously. Furtherit may be a naked antibody or conjugated, e.g., to a cytotoxic agent. Inanother preferred aspect of this method, the antibody is administered nomore than about once every other week, more preferably about once amonth, to the subject.

The invention also provides a composition comprising the antibody of anyof the preceding embodiments and a carrier, such as a pharmaceuticallyacceptable carrier.

Another aspect of the invention is an isolated nucleic acid encoding anantibody of any one of the preceding embodiments. Expression vectorscomprising such nucleic acid, and those encoding the antibodies of theinvention, are also provided. Also provided is a host cell comprising anucleic acid encoding an antibody of the invention. Any of a variety ofhost cells can be used. In one embodiment, the host cell is aprokaryotic cell, for example, E. coli. In another embodiment, the hostcell is a eukaryotic cell, for example a yeast cell or mammalian cellsuch as a Chinese Hamster Ovary (CHO) cell.

In another aspect, the invention provides methods for making an antibodyof the invention. For example, the invention provides a method of makingor producing an anti-LTα antibody (which, as defined herein includesfull length and fragments thereof, provided they contain an Fc region),said method comprising culturing a suitable host cell comprising anucleic acid encoding an antibody of the invention (preferablycomprising a recombinant vector of the invention encoding said antibody(or fragment thereof)), under conditions to produce the antibody, andrecovering said antibody. The antibody may be recovered from the hostcell or host cell culture. In a preferred embodiment, the antibody is anaked antibody. In another preferred embodiment, the antibody isconjugated with another molecule, the other molecule preferably being acytotoxic agent.

Still another aspect of the invention is an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises an antibody of any of the preceding embodimentsand a package insert indicating that the composition can be used totreat the indication the antibody is intended for, such as an autoimmunedisorder. A second medicament, as noted above, may be added to sucharticle, for example, in a separate container, in addition to theantibody, which is the first medicament.

In a further aspect, the invention encompasses a hybridoma deposited atthe ATCC on Apr. 19, 2006 under Deposit No. PTA-7538. Further providedis an antibody secreted by such a hybridoma.

If the autoimmune disease is RA specifically, one aspect is a methodwherein the patient has never been previously administered a medicamentfor the RA. An alternative aspect is a method wherein the patient hasbeen previously administered at least one medicament for the RA, morepreferably wherein the patient was not responsive to at least onemedicament that was previously administered. The previously administeredmedicament or medicaments to which the patient may be non-responsiveinclude an immunosuppressive agent, cytokine antagonist, integrinantagonist, corticosteroid, analgesic, DMARD, NSAID, or CD20 antagonist,and more preferably an immunosuppressive agent, cytokine antagonist,integrin antagonist, corticosteroid, DMARD, NSAID, or CD20 antagonist,such as a CD20 antibody, which is preferably not rituximab or ahumanized 2H7. Most preferably, such previously administeredmedicament(s) are a DMARD or a TNF inhibitor such as anti-TNF-alphaantibody or TNF-Ig.

In another preferred aspect of the invention for using the antibodiesherein to treat an autoimmune disease such as RA, the patient hasexhibited an inadequate response to one or more TNF inhibitors or to oneor more DMARDs. More preferably, the RA is early RA or incipient RA.

In another embodiment, a method is provided wherein at least about threemonths after the administration of the antibody herein to a patient withRA or joint damage, an imaging test is given that measures a reductionin bone or soft tissue joint damage as compared to baseline prior to theadministration, and the amount of the antibody administered is effectivein achieving a reduction in the joint damage. In a preferred aspect, thetest measures a total modified Sharp score. In a preferred aspect, themethod further comprises an additional administration to the patient ofthe antibody herein in an amount effective to achieve a continued ormaintained reduction in joint damage as compared to the effect of aprior administration of the antibody. In a further aspect, the antibodyherein is additionally administered to the patient even if there is noclinical improvement in the patient at the time of the testing after aprior administration. The clinical improvement is preferably determinedby assessing the number of tender or swollen joints, conducting a globalclinical assessment of the patient, assessing erythrocyte sedimentationrate, assessing the amount of C-reactive protein level, or usingcomposite measures of disease activity.

Preferred second medicaments for treatment of RA and joint damageinclude an immunosuppressive agent, a DMARD, a pain-control agent, anintegrin antagonist, a NSAID, a cytokine antagonist, a bisphosphonate,an antagonist to a B-cell surface marker such as, for example, BR3-Fc,BR3 antibody, CD20 antibody, CD40 antibody, CD22 antibody, CD23antibody, or a combination thereof. If the second medicament is a DMARD,preferably it is selected from the group consisting of auranofin,chloroquine, D-penicillamine, injectable gold, oral gold,hydroxychloroquine, sulfasalazine, myocrisin, and methotrexate. If thesecond medicament is a NSAID, preferably it is selected from the groupconsisting of: fenbufen, naprosyn, diclofenac, etodolac, indomethacin,aspirin and ibuprofen. If the second medicament is an immunosuppressiveagent, it is preferably selected from the group consisting ofetanercept, infliximab, adalimumab, leflunomide, anakinra, azathioprine,and cyclophosphamide. Other groups of preferred second medicamentsinclude anti-alpha4, etanercept, infliximab, etanercept, adalimumab,kinaret, efalizumab, osteoprotegerin (OPG), anti-receptor activator ofNFκB ligand (anti-RANKL), anti-receptor activator of NFκB-Fc (RANK-Fc),pamidronate, alendronate, actonel, zolendronate, clodronate,methotrexate, azulfidine, hydroxychloroquine, doxycycline, leflunomide,sulfasalazine (SSZ), prednisolone, interleukin-1 receptor antagonist,prednisone, or methylprednisolone. Another preferred group of secondmedicaments includes infliximab, an infliximab/methotrexate (MTX)combination, MTX, etanercept, a corticosteroid, cyclosporin A,azathioprine, auranofin, hydroxychloroquine (HCQ), combination ofprednisolone, MTX, and SSZ, combinations of MTX, SSZ, and HCQ, thecombination of cyclophosphamide, azathioprine, and HCQ, and thecombination of adalimumab with MTX More preferably, the corticosteroidis prednisone, prednisolone, methylprednisolone, hydrocortisone, ordexamethasone. In another preferred embodiment, the second medicament isMTX, which is more preferably administered perorally or parenterally.

In another aspect, the treatment methods herein further comprisere-treating the patient by re-administering an effective amount of theantibody herein to the patient. Preferably, the re-treatment iscommenced at least about 24 weeks after the first administration of theantibody. In another embodiment, a second re-treatment is commenced, andmore preferably wherein the second re-treatment is commenced at leastabout 24 weeks after the second administration of the antibody. Inanother embodiment, where the autoimmune disease is RA, joint damage hasbeen reduced after the re-treatment. In an alternative embodiment, noclinical improvement is observed in the patient at the time of thetesting after the re-treatment, especially wherein the clinicalimprovement is determined by assessing the number of tender or swollenjoints, conducting a global clinical assessment of the patient,assessing erythrocyte sedimentation rate, assessing the amount ofC-reactive protein level, or using composite measures of diseaseactivity.

In another aspect, the invention herein provides a method foradvertising an antibody herein or a pharmaceutically acceptablecomposition thereof comprising promoting, to a target audience, the useof the antibody or pharmaceutical composition thereof for treating apatient or patient population exhibiting an autoimmune disease such asRA, MS, lupus, or an IBD.

In another aspect, the invention provides an article of manufacturecomprising, packaged together, a pharmaceutical composition comprisingan antibody herein and a pharmaceutically acceptable carrier and a labelstating that the antibody or pharmaceutical composition is indicated fortreating patients with an autoimmune disease such as RA, MS, lupus, oran IBD. In a preferred embodiment, the article further comprises acontainer comprising a second medicament, wherein the antibody herein isa first medicament, further comprising instructions on the packageinsert for treating the patient with an effective amount of the secondmedicament, which is preferably methotrexate.

In a still further aspect, the invention provides a method for packagingan antibody herein or a pharmaceutical composition of the antibodycomprising combining in a package the antibody or pharmaceuticalcomposition and a label stating that the antibody or pharmaceuticalcomposition is indicated for treating patients exhibiting an autoimmunedisease such as RA, MS, lupus, or an IBD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the full-length sequences for the 2C8 chimera light andheavy chains (SEQ ID NOS:32 and 33, respectively).

FIG. 1B depicts the full-length sequences for the 3F12 chimera light andheavy chains (SEQ ID NOS:34 and 35, respectively), as well as the 3F12chimera heavy-chain variable region (SEQ ID NO:31).

FIG. 1C depicts the sequence of the light-chain variable region for animproved 3F12 chimera (referred to as chimera2 in the examples below)(3F12.2D3) (SEQ ID NO:36) and the sequence of the light-chain constantregion of 3F12.2D3 (SEQ ID NO:37). The bold type of the residuesindicates those identified from various chemical/enzymatic cleavages.

FIG. 1D depicts the sequence of the heavy-chain variable region for animproved 3F12 chimera (3F12.2D3) (SEQ ID NO:38) and the sequence of theheavy-chain constant region of 3F12.2D3 (SEQ ID NO:39). The bold type ofthe residues indicates those identified from various chemical/enzymaticcleavages, and the asterisk indicates a glycosylation site.

FIG. 2A depicts alignment of sequences of the variable light chain forthe following: chimeric 2C8 antibody (SEQ ID NO:23), humanized 2C8.v2(SEQ ID NO: 24), and human kappa subgroup I consensus sequence (SEQ IDNO:40). The CDR sequences of each are shown in brackets, and asterisksare used to show differences among the aligned sequence amino acidresidues.

FIG. 2B depicts alignment of sequences of the variable heavy chain forthe following: chimeric 2C8 antibody (SEQ ID NO:25), humanized 2C8.v2(SEQ ID NO:26), and human subgroup III consensus sequence (SEQ IDNO:41). The CDR sequences of each are shown in brackets, and asterisksare used to show differences among the aligned sequence amino acidresidues.

FIG. 2C depicts the light- and heavy-chain sequences for versions 2C8.v7and2C8.v12 (SEQ ID NOS:42-45).

FIG. 3A depicts alignment of sequences of the variable light chain forthe following: chimeric 3F12 antibody (SEQ ID NO:27), humanized 3F12.v5(SEQ ID NO:28), and human kappa subgroup I consensus sequence (SEQ IDNO:40). The CDR sequences of each are shown in brackets, and asterisksare used to show differences among the aligned sequence amino acidresidues.

FIG. 3B depicts alignment of sequences of the variable heavy chain forthe following: original chimeric 3F12 antibody (not 3F12.2D3) (SEQ IDNO:29), humanized 3F12.v5 (SEQ ID NO:30), and human subgroup IIIconsensus sequence (SEQ ID NO:41). The CDR sequences of each are shownin brackets, and asterisks are used to show differences among thealigned sequence amino acid residues.

FIGS. 4A and 4B show that hamster-murine chimeric anti-LTα antibody S5H3inhibits antibody-induced arthritis (AIA). FIG. 4A shows the results ofpreventative use of the anti-LTα antibody versus a Fc DANA mutation(anti-LT.Fcmutant) and a TNFRII.Fc mutant (a TNFR.Ig immunoadhesin) aswell as a control isotype (anti-ragweed IgG2a monoclonal antibody). FIG.4B shows that the anti-LTα antibody inhibited development of arthritisin an AIA model when administered therapeutically, versus controlisotype and TNFRII.Fc mutant.

FIGS. 5A and 5B show that hamster-murine chimeric anti-LTα antibody S5H3inhibits collagen-induced arthritis (CIA). FIG. 5A shows that S5H3inhibited arthritis in a CIA-preventative model, versus a Fc DANAmutation (anti-LT.Fc mutant) as well as a control isotype (anti-ragweedIgG2a monoclonal antibody) and was comparable to a TNFRII.Fc mutant.FIG. 5B shows that the S5H3 anti-LTα antibody inhibited development ofarthritis in a CIA model when administered therapeutically, versuscontrol isotype, and was comparable to the TNFRII.Fc mutant.

FIG. 6 shows that hamster-murine chimeric anti-LTα antibody S5H3 delaysonset and severity of EAE disease in MBP-TCR transgenic mice, versus acontrol isotype (anti-gp120 IgG1 monoclonal antibody).

FIGS. 7A, 7B, and 7C show that treatment with hamster-murine chimericanti-LTα antibody S5H3 does not affect the T-cell-dependent anti-TNPIgM, IgG1, and IgG2a responses, respectively, as compared to isotypecontrol (anti-ragweed), anti-LT.Fc mutant, and CTLA4.Fc (a positivecontrol).

FIG. 8 shows that treatment with hamster-murine chimeric anti-LTαantibody S5H3 does not affect T-cell-independent antibody (IgM, IgG1,IgG2a, and IgG3) responses relative to isotype control (anti-ragweed)and TNFRII.Fc mutant.

FIG. 9 shows that anti-LTα chimeric monoclonal antibody 2C8 preventsGVHD in human SCID mice as compared to the DANA anti-LTα 2C8 Fc mutantand the isotype control (trastuzumab, human IgG1). CTLA4.Fc mutant wasalso used as a positive control.

FIG. 10A shows that chimeric anti-LTα antibodies 2C8 and 3F12 bind tohuman LTα3.

FIG. 10B shows that hamster-murine chimeric anti-LTα antibody S5H3 aswell as anti-LT.Fc mutant bind to murine LTα3.

FIG. 11A shows through enzyme-linked immunosorbent assay (ELISA) resultsthat hamster-murine chimeric anti-LTα S5H3 antibody binds to murineLTα1β2 complex, as does the anti-LT.Fc mutant.

FIG. 11B shows FACS assay results for binding of chimeric anti-LTαantibodies 3F12 and 2C8 to the LTα on the surface of human cellscomplexed with LTβ. They are compared to LTβ-receptor.huIgG1 and isotypecontrol huIgG1 (trastuzumab).

FIG. 11C shows FACS assay results for binding of hamster-murine chimericanti-LTα antibody S5H3 to the LTα on the surface of murine cellscomplexed with LTβ. It is compared to LTβ-receptor murine IgG2a, anti-LTS5H3Fc mutant, and isotype control (anti-IL122 muIgG2a).

FIGS. 12A and 12B show that chimeric anti-LTα antibodies 2C8 and 3F12bind to human Th1 and Th2 cells, respectively, and show binding resultsof LThR.Fc, TNFRII.Fc, and isotype control for comparison.

FIG. 12C-12E show that anti-LTα humanized antibody 2C8.vX does not bindto resting cells (FIG. 12C), but does bind to human Th1 and Th17 cells(FIGS. 12D and E, respectively), with a comparison of its binding withthat of isotype control.

FIG. 13A shows unactivated T cells treated with LTbR.Fc (solid line),chimeric anti-LTα 3F12 antibody (dashed line), and isotype control(shaded area).

FIG. 13B shows anti-CD3- and anti-CD28-activated CD4 T cells treatedwith the reagents of FIG. 13A.

FIG. 13C shows anti-CD3- and anti-CD28-activated CD8 T cells treatedwith the reagents of FIG. 13A.

FIG. 13D shows IL15-activated CD56 NK cells treated with the reagents ofFIG. 13A.

FIG. 13E shows IgM- and CD40L-activated CD19 B cells treated with thereagents of FIG. 13A.

FIG. 14A shows that chimeric anti-LTα antibodies 2C8 and 3F12functionally block human LTα3 versus the isotype control (trastuzumab).

FIG. 14B shows that hamster-murine chimeric anti-LTα antibody S5H3 andthe anti-LT.Fc mutant functionally block murine LTα3 versus the isotypecontrol (trastuzumab).

FIG. 14C shows that chimeric anti-LTα antibodies 2C8 and 3F12 blockLTα3-induced IL-8 in A549 cells versus the isotype control(trastuzumab).

FIG. 14D shows that chimeric anti-LTα antibodies 2C8 and 3F12, as wellas TNFRII.Fc mutant, block LTα3-induced ICAM expression on HUVEC versusisotype control (trastuzumab). The response of unstimulated cells isalso shown.

FIG. 14E shows that chimeric anti-LTα antibody 2C8 blocks LTα3-inducedNFkB activation, versus the isotype control. The response ofunstimulated cells is also shown.

FIG. 15A shows that chimeric anti-LTα antibody 2C8 functionally blocksLTα1β2-induced NFkB activation versus the isotype control. The responseof unstimulated cells is also shown.

FIGS. 15B, 15C, and 15D show that chimeric anti-LTα antibody 2C8 blocks,respectively, LTα3-, LTα2β1-, and LTα1β2-induced cytotoxicity.

FIG. 15E shows that chimeric anti-LTα antibodies 2C8 and 3F12 blockLTα1β2-induced ICAM expression on HUVEC, versus the isotype control. Theresponse of unstimulated cells is also shown.

FIG. 16 shows that chimeric anti-LTα antibody 2C8 can killLTαβ-expressing cells, versus anti-LTα 2C8 Fc mutant.

FIG. 17A-H show that chimeric anti-LTα antibody 2C8 blocks cytokine andchemokine secretion in HUVEC cells, with FIGS. 17A-17D respectivelyshowing KC/IL-8, RANTES, IP10, and IL-6 with LTα1β2, and FIGS. 17E-17Hrespectively showing KC/IL-8, RANTES, IP10, and IL-6 with LTα3 versuscontrol.

FIG. 17I-P show that hamster-murine chimeric anti-LTα antibody S5H3blocks cytokine and chemokine secretion in 3T3 cells, with FIGS. 17I-17Lrespectively showing KC, RANTES, IP10, and IL-6 with LTα1β2, and FIGS.17M-17P respectively showing KC, RANTES, IP10, and IL-6 with LTα3 versuscontrol.

FIG. 18A depicts alignment of sequences of the variable light chain forthe following: chimeric 2C8 antibody (SEQ ID NO:23), humanized 2C8.vX(SEQ ID NO:102), and human kappa subgroup I consensus sequence (SEQ IDNO:40). The CDR sequences of each are shown in brackets, and asterisksare used to show differences among the aligned sequence amino acidresidues.

FIG. 18B depicts alignment of sequences of the variable heavy chain forthe following: chimeric 2C8 antibody (SEQ ID NO:25), humanized 2C8.vX(SEQ ID NO:103), and human subgroup III consensus sequence (SEQ IDNO:41). The CDR sequences of each are shown in brackets, and asterisksare used to show differences among the aligned sequence amino acidresidues.

FIGS. 19A and 19B show, respectively, ELISAs of LTα3 and LTα1β2 bindingof 2C8.vX antibodies in comparison to TNFRII.Fc/isotype control.

FIGS. 20A and 20B show, respectively, blocking of human LTα3 and LTα1β2by 2C8vX antibodies in comparison to isotype control/TNFRII.Fc using anassay for testing blocking of human LT-induced cytotoxicity.

FIG. 21 provides a comparison of how various molecules blocked LTα3 in afunctional assay showing inhibition of ICAM1 upregulation by LTα3 onHUVEC cells. Specifically, isotype, 2C8.vX and TNFRII.Fc were compared.

FIG. 22 provides a comparison of how various molecules blocked LTα1β2 ina functional assay showing LTα1β2 blocking using 293-LTβR cells.Specifically, isotype and 2C8.vX were compared along with unstimulatedcells.

FIG. 23 shows ADCC activity of 2C8.vX using a protocol involving293-LTα1β2 cells. 2C8.vX was compared with the 2C8.vX DANA mutant (Fcmutant) and isotype control.

FIG. 24 shows GVHD survival results with 2C8.vX and CTLA4-Fc, ascompared to controls (2C8.vX DANA Fc mutant and isotype control), in ahuman SCID model.

FIG. 25A-D show that the 2C8.vX antibody depletes LTα1β2-expressing Tand B cells in the human SCID GVHD model. The results in FIG. 25A showtotal positive human cells at day 2 for isotype control, 2C8.vX, and2C8.vX.DANA mutant. FIG. 25B-D show the results of gating using the CD4,CD8, and CD19 antibodies, respectively.

FIG. 26A depicts alignment of sequences of the variable light chain forthe following: chimeric 3F12 antibody (SEQ ID NO:27), humanized 3F12.v14(SEQ ID NO:108), and human kappa subgroup I consensus sequence (SEQ IDNO:40). The CDR sequences of each are shown in brackets, and asterisksare used to show differences among the aligned sequence amino acidresidues.

FIG. 26B depicts alignment of sequences of the variable heavy chain forthe following: chimeric 3F12 antibody (SEQ ID NO:29), humanized 3F12.v14(SEQ ID NO:109), and human subgroup III consensus sequence (SEQ IDNO:41). The CDR sequences of each are shown in brackets, and asterisksare used to show differences among the aligned sequence amino acidresidues.

FIG. 27 shows ADCC activity of 2C8.vX and its afucosylated counterpart,wherein AF=afucosylated antibody and WT=regular CHO cell-line productionantibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture(R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press,Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al.,eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction,(Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning(Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas etal., 2001).

Definitions

“Lymphotoxin-α or “LTα” is defined herein as a biologically activepolypeptide having the amino acid sequence shown in FIG. 2A of U.S. Pat.No. 5,824,509. LTα is defined to specifically exclude human TNFα or itsnatural animal analogues (Pennica et al., Nature 312:20/27: 724-729(1984) and Aggarwal et al., J. Biol. Chem. 260: 2345-2354 (1985)). LTαis defined to specifically exclude human LTβ as defined, for example, inU.S. Pat. No. 5,661,004.

“Lymphotoxin-α3 trimer” or “LTα3” refers to a homotrimer of LTαmonomers. This homotrimer is anchored to the cell surface by the LTβ,transmembrane and cytoplasmic domains.

“Lymphotoxin-αβ” or “LTαβ” or “LTαβ complex” refers to a heterotrimer ofLTα with LTβ. These heterotrimers contain either two subunits of LTα andone subunit of LTβ (LTα2β1), or one subunit of LTα and two of LTβ(LTα1β2).

“Tumor necrosis factor receptor-I” or “TNFRI” and “tumor necrosis factorreceptor-II” or “TNFRII” refer to cell-surface TNF receptors for theLTαβ homotrimer, also known as p55 and p75, respectively.

“Lymphotoxin-β receptor” or “LTβ-R” refers to the receptor to which theLTαβ heterotrimers bind.

“Regulatory cytokines” are cytokines the abnormal levels of whichindicate the presence of an autoimmune disorder in a patient. Suchcytokines include, for example, interleukin-1 (IL-1), IL-2, IL-3, IL-4,IL-5, IL-69 IL-7, IL-8, IL-10, IL-12, IL-13, IL-14, IL-15, IL-18, IL-23,IL-24, IL-25, IL-26, BLyS/April, TGF-α, TGF-β, interferon-α (IFN-α),IFN-β, IFN-γ, MIP-1, MIF, MCP-1, M-CSF or G-CSF, a lymphotoxin, LIGHT,4-1BB ligand, CD27 ligand, CD30 ligand, CD40 ligand, Fas ligand, GITRligand, OX40 ligand, RNAK ligand, THANK, TRAIL, TWEAK and VEG1. Thisgroup includes TNF family members, which include but are not limited to,TNF-α, LTs such as LTα, LTβ, and LIGHT. For a review of the TNFsuperfamily, see MacEwan, Br. J. Pharmacology 135: 855-875 (2002).Preferably, the regulatory cytokine is an IL such as IL-1b or IL-6and/or a TNF family member.

“Inflammatory cytokines associated with rheumatoid arthritis” refer toIL-6, IL-1b, and TNFα, associated with RA pathology, which can beinhibited systemically and or in the joints in an in vitrocollagen-induced arthritis assay.

“LTαβ-expressing cells” are cells that express or secrete the LTαβheterotrimers.

The expression “modulates LTαβ-expressing cells” refers to depleting oraltering proteins made by the cells such as cytokines, chemokines, orgrowth factors, with the cells including, for example, monocytes,dendritic cells, T cells, and B cells.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full-lengthor intact monoclonal antibodies), polyclonal antibodies, multivalentantibodies, and multispecific antibodies (e.g., bispecific antibodies solong as they exhibit the desired biological activity), and may alsoinclude certain antibody fragments (as described in greater detailherein). An antibody can be chimeric, human, humanized, and/or affinitymatured.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, more preferablymost or all, of the functions normally associated with that portion whenpresent in an intact antibody. In one embodiment, an antibody fragmentcomprises an antigen-binding site of the intact antibody and thusretains the ability to bind antigen. In another embodiment, an antibodyfragment, for example one that comprises the Fc region, retains at leastone of the biological functions normally associated with the Fc regionwhen present in an intact antibody, such as FcRn binding, antibodyhalf-life modulation, ADCC function, and complement binding. In oneembodiment, an antibody fragment is a monovalent antibody that has an invivo half life substantially similar to an intact antibody. For example,such an antibody fragment may comprise an antigen binding arm linked toan Fc sequence capable of conferring in vivo stability to the fragment.In one embodiment, an antibody of the invention is a one-armed antibodyas described in WO 2005/063816, for example, an antibody comprising Fcmutations constituting “knobs” and “holes”. For example, a hole mutationcan be one or more of T366A, L368A and/or Y407V in an Fc polypeptide,and a cavity mutation can be T366W. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

For the purposes herein, an “intact antibody” is one comprising heavyand light variable domains as well as an Fc region.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they may be synthesized uncontaminated by otherantibodies. The modifier “monoclonal” is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies useful in the present invention maybe prepared by the hybridoma methodology first described by Kohler etal., Nature, 256:495 (1975), or may be made using recombinant DNAmethods in, e.g., bacterial, non-animal eukaryotic, animal, or plantcells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies”may also be isolated from phage antibody libraries using the techniquesdescribed in, e.g., Clackson et al., Nature, 352:624-628 (1991) andMarks et al., J. Mol. Biol., 222:581-597 (1991).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Methods of makingchimeric antibodies are known in the art.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)that contain minimal sequence derived from non-human immunoglobulin. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a CDR of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv FR residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and maximize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence, althoughthe FR regions may include one or more amino acid substitutions thatimprove binding affinity. The number of these amino acid substitutionsin the FR is typically no more than six in the H chain, and no more thanthree in the L chain. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321:522-525 (1986); Reichmann et al., Nature,332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596(1992). See also Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038(1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994). Thehumanized antibody includes a PRIMATIZED® antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest. Methods of making humanized antibodies are known in the art.

A “human antibody” is one that possesses an amino acid sequence thatcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can also be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991).

An “affinity-matured” antibody is an antibody with one or morealterations in one or more CDRs thereof that result in an improvement inthe affinity of the antibody for antigen, compared to a parent antibodythat does not possess those alteration(s). Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity-matured antibodies are produced by proceduresknown in the art. Marks et al. Bio/Technology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by: Barbas etal., Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J.Mol. Biol. 226:889-896 (1992).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten, or another naturally occurring or syntheticcompound. Preferably, the target antigen is α polypeptide. An “acceptorhuman framework” for the purposes herein is a framework comprising theamino acid sequence of a VL or VH framework derived from a humanimmunoglobulin framework, or from a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor human consensus framework may comprise the same amino acid sequencethereof, or may contain pre-existing amino acid sequence changes. Wherepre-existing amino acid changes are present, preferably no more thanfive, and more preferably four or less, and still more preferably threeor less, pre-existing amino acid changes are present. Where pre-existingamino acid changes are present in a VH, preferably those changes areonly at three, two, or one of positions 71, 73, and 78; for instance,the histidine residues at those positions may be alanine residues. Inone embodiment, the VL acceptor human framework is identical in sequenceto the VL human immunoglobulin framework sequence or human consensusframework sequence.

A “human consensus framework” is a framework that represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Typically, this subgroup of sequences is a subgroup asin Kabat et al., supra. In one embodiment, for the VL, the subgroup issubgroup kappa I as in Kabat et al., supra. In one embodiment, for theVH, the subgroup is subgroup III as in Kabat et al, supra.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 46)-H1-WVRQAPGKGLEWVG (SEQ ID NO: 47)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 48)-H3-WGQGTLVTVSS (SEQ ID NO: 49),where N is any amino acid residue.

In another embodiment, the VH subgroup III consensus framework aminoacid sequence comprises at least a portion or all of each of thefollowing sequences:

EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 46)-H1-WVRQAPGKGLEWVG (SEQ ID NO: 47)-H2-RATFSADNSKNTAYLQMNSLRAEDTAVYYCAD (SEQ ID NO: 50)-H3-WGQGTLVTVSS (SEQ ID NO: 49),where N is any amino acid residue.

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VL subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 51) DIQMTQSPSSLSASVGDRVTITC- (SEQ ID NO: 52)L1-WYQQKPGKAPKLQIY- (SEQ ID NO: 53) L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC-(SEQ ID NO: 54) L3-FGQGTKVEIKR.

In another embodiment, the VL subgroup I consensus framework amino acidsequence comprises at least a portion or all of each of the followingsequences:

(SEQ ID NO: 51) DIQMTQSPSSLSASVGDRVTITC- (SEQ ID NO: 55)L1-WYQQKPGKAPKLLIY- (SEQ ID NO: 53) L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC-(SEQ ID NO: 54) L3-FGQGTKVEIKR.

The term “complementarity-determining region”, or “CDR”, when usedherein refers to the regions of an antibody variable domain that arehypervariable in sequence and/or form structurally defined loops.Generally, antibodies comprise six hypervariable regions; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of CDRdelineations are in use and are encompassed herein. The Kabat CDRs arebased on sequence variability and are the most commonly used (Kabat etal., supra). Chothia refers instead to the location of the structuralloops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbMhypervariable regions represent a compromise between the Kabat CDRs andChothia structural loops, and are used by Oxford Molecular's AbMantibody modeling software. The “contact” hypervariable regions arebased on an analysis of the available complex crystal structures. Theresidues from each of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L24-L32 L30-L36 L2L50-L56 L50-L56 L50-L56 L46-L55 L3 L89-L97 L89-L97 L89-L97 L89-L96 H1H31-H35B H26-H32B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H52-H56 H47-H58H3 H95-H102 H95-H102 H95-H102 H93-H101

CDRs may comprise “extended CDRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2), and 89-97 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65(H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domainresidues are numbered according to Kabat et al., supra, for each ofthese definitions. Throughout the present specification and claims, thenumbering of the residues in an immunoglobulin heavy chain is that ofthe EU index as in Kabat et al., supra. The “EU index as in Kabat”refers to the residue numbering of the human IgG1 EU antibody.

An “unmodified human framework” is a human framework that has the sameamino acid sequence as the acceptor human framework, e.g. lacking humanto non-human amino acid substitution(s) in the acceptor human framework.

An “altered CDR” for the purposes herein is a CDR comprising one or more(e.g. one to about 16) amino acid substitution(s) therein.

An “un-modified CDR” for the purposes herein is a CDR having the sameamino acid sequence as a non-human antibody from which it was derived,i.e. one lacking one or more amino acid substitutions.

“Framework” or “FR” residues are those variable domain residues otherthan the CDR residues as herein defined.

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody that mimics atleast one of the functional activities of a polypeptide of interest.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably to more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “variable-domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy-chain variable domains or light-chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard”Kabat-numbered sequence.

A “parent polypeptide” is a polypeptide comprising an amino acidsequence that lacks one or more of the Fc-region modifications disclosedherein and that differs in effector function compared to a polypeptidevariant as herein disclosed. The parent polypeptide may comprise anative-sequence Fc region or an Fc region with pre-existing amino acidsequence modifications (such as additions, deletions, and/orsubstitutions).

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native-sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy-chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheFc region of an immunoglobulin generally comprises two constant domains,CH2 and CH3. The last residue, lysine, in the heavy chain of IgG1 can,but does not have to, be present as the terminal residue in the Fc inthe mature protein.

The “CH2 domain” of a human IgG Fc region (also referred to as “Cγ2”domain) usually extends from about amino acid 231 to about amino acid340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec. Immunol. 22: 161-206 (1985).

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.e. from about amino acid residue 341 to aboutamino acid residue 447 of an IgG)

A “functional Fc region” possesses an “effector function” of anative-sequence Fc region. Exemplary “effector functions” include C1qbinding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulationof cell-surface receptors (e.g. LT receptor), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g. an antibody variable domain) and can be assessed usingvarious assays as herein disclosed, for example.

A “native-sequence Fc region” or “wild-type Fc region” comprises anamino acid sequence identical to the amino acid sequence of an Fc regionfound in nature. Native-sequence human Fc regions are known in the artand include a native-sequence human IgG1 Fc region (non-A and Aallotypes); native-sequence human IgG2 Fc region; native-sequence humanIgG3 Fc region; and native-sequence human IgG4 Fc region, as well asnaturally occurring allelic variants thereof.

A “variant Fc region” comprises an amino acid sequence that differs fromthat of a native-sequence Fc region by virtue of at least one “aminoacid modification” as herein defined. Preferably, the variant Fc regionhas at least one amino acid substitution compared to a native-sequenceFc region or to the Fc region of a parent polypeptide, e.g. from aboutone to about ten amino acid substitutions, and preferably from about oneto about five amino acid substitutions in a native-sequence Fc region orin the Fc region of the parent polypeptide. The variant Fc region hereinwill preferably possess at least about 80% homology with anative-sequence Fc region and/or with an Fc region of a parentpolypeptide, more preferably at least about 90% homology therewith, andmost preferably at least about 95% homology therewith.

The term “Fc region-containing polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions herein), whichcomprises an Fc region.

The terms “Fc receptor” and “FcR” are used to describe a receptor thatbinds to the Fc region of an IgG antibody. The preferred FcR is anative-sequence human FcR. In one embodiment, the FcR is a FcγR thatincludes receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain (see review in Daëron, Annu. Rev.Immunol. 15:203-234 (1997)). The term includes allotypes, such asFcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131,and/or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994);and de Haas et al., J. Lab. Clin. Med., 126:330-341 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., Eur. J.Immunol. 24:2429-2434 (1994)).

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoieticcells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu.Rev. Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMCs) and NKcells. Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocytesthat mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells,and neutrophils; with PBMCs and NK cells being preferred. The effectorcells may be isolated from a native source thereof, e.g. from blood orPBMCs as described herein.

“Complement-dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)that are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

A polypeptide with a variant IgG Fc having “altered” FcR bindingaffinity or ADCC activity is one that has either enhanced or diminishedFcR binding activity (FcγR or FcRn) and/or ADCC activity compared to aparent polypeptide or to a polypeptide comprising a native-sequence Fcregion. The variant Fc that “exhibits increased binding” to an FcR bindsat least one FcR with better affinity than the parent polypeptide. Theimprovement in binding compared to a parent polypeptide may be aboutthree-fold, preferably about 5-, 10-, 25-, 50-, 60-, 100-, 150-, 200-,and up to 500-fold, or about 25% to 1000% improvement in binding. Thepolypeptide variant that “exhibits decreased binding” to an FcR binds atleast one FcR with less affinity than a parent polypeptide. The decreasein binding compared to a parent polypeptide may be about 40% or moredecrease in binding. Such Fc variants that display decreased binding toan FcR may possess little or no appreciable binding to an FcR, e.g.,about 0-20% binding to the FcR compared to a native-sequence IgG Fcregion.

The polypeptide having a variant Fc that binds an FcR with “betteraffinity” or “higher affinity” than a polypeptide or parent polypeptidehaving wild-type or native-sequence IgG Fc is one that binds any one ormore of the above identified FcRs with substantially better bindingaffinity than the parent polypeptide with native-sequence Fc, when theamounts of polypeptide with variant Fc and parent polypeptide in thebinding assay are essentially the same. For example, the variant Fcpolypeptide with improved FcR binding affinity may display from abouttwo-fold to about 300-fold, preferably, from about three-fold to about170-fold, improvement in FcR binding affinity compared to the parentpolypeptide, where FcR binding affinity is determined as known in theart.

The polypeptide comprising a variant Fc region that “exhibits increasedADCC” or mediates ADCC in the presence of human effector cells moreeffectively than a polypeptide having wild-type IgG Fc is one that invitro or in vivo is substantially more effective at mediating ADCC, whenthe amounts of polypeptide with variant Fc region and the polypeptidewith wild-type Fc region used in the assay are essentially the same.Generally, such variants will be identified using the in vitro ADCCassay as herein disclosed, but other assays or methods for determiningADCC activity, e.g. in an animal model, etc, are contemplated. Thepreferred variant is from about five-fold to about 100-fold, morepreferably from about 25- to about 50-fold, more effective at mediatingADCC than the wild-type Fc.

An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitution, insertion, and/or deletion. Thepreferred amino acid modification herein is a substitution.

An “amino acid modification at” a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent to thespecified residue. By insertion “adjacent to” a specified residue ismeant insertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e., encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Preferably, the replacement residue is not cysteine. Substitutionwith one or more non-naturally occurring amino acid residues is alsoencompassed by the definition of an amino acid substitution herein. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoserine, and otheramino acid residue analogues such as those described in Eliman et al.,Meth. Enzym. 202:301-336 (1991). For generation of such non-naturallyoccurring amino acid residues, the procedures of Noren et al., Science244:182 (1989) and Eliman et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor tRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

The term “conservative” amino acid substitution as used within thisinvention is meant to refer to amino acid substitutions that substitutefunctionally equivalent amino acids. Conservative amino acid changesresult in silent changes in the amino acid sequence of the resultingpolypeptide. For example, one or more amino acids of a similar polarityact as functional equivalents and result in a silent alteration withinthe amino acid sequence of the polypeptide. In general, substitutionswithin a group may be considered conservative with respect to structureand function. However, the skilled artisan will recognize that the roleof a particular residue is determined by its context within thethree-dimensional structure of the molecule in which it occurs. Forexample, Cys residues may occur in the oxidized (disulfide) form, whichis less polar than the reduced (thiol) form. The long aliphatic portionof the Arg side chain may constitute a critical feature of itsstructural or functional role, and this may be best conserved bysubstitution of a nonpolar, rather than another basic, residue. Also, itwill be recognized that side chains containing aromatic groups (Trp,Tyr, and Phe) can participate in ionic-aromatic or “cation-pi”interactions. In these cases, substitution of one of these side chainswith a member of the acidic or uncharged polar group may be conservativewith respect to structure and function. Residues such as Pro, Gly, andCys (disulfide form) can have direct effects on the main-chainconformation, and often may not be substituted without structuraldistortions.

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence. While the insertionwill usually consist of the insertion of one or two amino acid residues,the present application contemplates larger “peptide insertions”, e.g.,insertion of about three to about five or even up to about ten aminoacid residues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

Amino acids may be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (O)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

The “hinge region” is generally defined as stretching from Glu216 toPro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). Hingeregions of other IgG isotypes may be aligned with the IgG1 sequence byplacing the first and last cysteine residues forming inter-heavy chainS—S bonds in the same positions.

The “lower hinge region” of an Fc region is normally defined as thestretch of residues immediately C-terminal to the hinge region, i.e.,residues 233 to 239 of the Fc region.

“C1q” is a polypeptide that includes a binding site for the Fc region ofan immunoglobulin. C1q together with two serine proteases, C1r and C1s,forms the complex C1, the first component of the CDC pathway. Human C1qcan be purchased commercially from, e.g. Quidel, San Diego, Calif.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g., the α chainthereof) that is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR α chain.

“Functional fragments” of the antibodies of the invention comprise aportion of an intact antibody, generally including the antigen-bindingor variable region of the intact antibody or the Fc region of anantibody that retains FcR binding capability. Examples of antibodyfragments include linear antibodies, single-chain antibody molecules,and multispecific antibodies formed from antibody fragments.

As used herein, the term “immunoadhesin” designates antibody-likemolecules that combine the binding specificity of a heterologous protein(an “adhesin”) with the effector functions of immunoglobulin constantdomains. Structurally, the immunoadhesins comprise a fusion of an aminoacid sequence with the desired binding specificity that is other thanthe antigen-recognition and binding site of an antibody (i.e., is“heterologous”), and an immunoglobulin constant-domain sequence. Theadhesin part of an immunoadhesin molecule typically is a contiguousamino acid sequence comprising at least the binding site of a receptoror a ligand. The immunoglobulin constant-domain sequence in theimmunoadhesin can be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD, or IgM. For example, useful immunoadhesins as second medicamentsherein include polypeptides that comprise the B-lymphocyte stimulator(BLyS) binding portions of a BLyS receptor without the transmembrane orcytoplasmic sequences of the BLyS receptor. In one embodiment, theextracellular domain of BR3 (BLyS receptor 3), TACI (transmembraneactivator and calcium-modulator and cyclophilin ligand interactor), orBCMA (B-cell maturation antigen) is fused to a constant domain of animmunoglobulin sequence.

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving two portions covalently linked together, where each of theportions is a polypeptide having a different property. The property maybe a biological property, such as activity in vitro or in vivo. Theproperty may also be a simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, etc. The twoportions may be linked directly by a single peptide bond or through apeptide linker containing one or more amino acid residues. Generally,the two portions and the linker will be in reading frame with eachother.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity that reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can be determined with the surface plasmonresonance technique using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) optical biosensor at 25° C. with immobilizedantigen CM5 chips at ˜10 response units (RU). Briefly, carboxymethylateddextran biosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of 1M ethanolamine to block unreacted groups, for kineticsmeasurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) areinjected in phosphate-buffered saline (PBS) with 0.05% TWEEN™ 20 (PBST)at 25° C. at a flow rate of approximately 25 μl/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIACORE® Evaluation Software version3.2™) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol293:865-881 (1999). However, if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate is preferablydetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The “Kd” or “Kd value” according to this invention is, in oneembodiment, measured by a radiolabeled antigen binding assay (RIA)performed with the Fab version of the antibody and antigen molecule asdescribed by the following assay that measures solution binding affinityof Fabs for antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen et al., J. Mol. Biol 293:865-881 (1999)). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (consistent with assessment of an anti-VEGF antibody, Fab-12,in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interestis then incubated overnight; however, the incubation may continue for alonger period (e.g., 65 hours) to insure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature for one hour. The solution is thenremoved and the plate washed eight times with 0.1% TWEEN™-20 in PBS.When the plates have dried, 150 μl/well of scintillant (MicroScint-20;Packard) is added, and the plates are counted on a TOPCOUNT™ gammacounter (Packard) for ten minutes. Concentrations of each Fab that giveless than or equal to 20% of maximal binding are chosen for use incompetitive binding assays.

The phrase “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, more preferably less thanabout 40%, still more preferably less than about 30%, even morepreferably less than about 20%, and most preferably less than about 10%as a function of the value for the reference/comparator antibody.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of statistical significance within the context ofthe biological characteristic measured by said values (e.g., Kd values,HAMA response). The difference between said two values is preferablygreater than about 10%, more preferably greater than about 20%, stillmore preferably greater than about 30%, even more preferably greaterthan about 40%, and most preferably greater than about 50% as a functionof the value for the reference/comparator antibody.

“Percent (%) amino acid sequence identity” and “homology” with respectto a peptide or polypeptide sequence are defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific peptide or polypeptide sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in theart can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are generated using thesequence comparison computer program ALIGN-2, authored by Genentech,Inc. The source code of ALIGN-2 has been filed with user documentationin the U.S. Copyright Office, Washington D.C., 20559, where it isregistered under U.S. Copyright Registration No. TXU510087. The ALIGN-2program is publicly available through Genentech, Inc., South SanFrancisco, Calif. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as describedherein using the ALIGN-2 computer program.

A “disorder” is any condition that would benefit from treatment with anantibody or method of the invention, regardless of mechanism, butincluding inhibiting or blocking the action of LTα3 or LTαβ and/or bydepleting LTα-positive cells. This condition includes, but is notlimited to, a medical condition or illness mediated by or related toelevated expression or activity, or abnormal activation, of LTα3 and/orLTαβ by any cell. This includes chronic and acute disorders such asthose pathological conditions that predispose the mammal to the disorderin question.

An “autoimmune disorder” herein is a disease or disorder arising fromand directed against an individual's own tissues or organs or aco-segregate or manifestation thereof or resulting condition therefrom.In many of these autoimmune and inflammatory disorders, a number ofclinical and laboratory markers may exist, including, but not limitedto, hypergammaglobulinemia, high levels of autoantibodies,antigen-antibody complex deposits in tissues, benefit fromcorticosteroid or immunosuppressive treatments, and lymphoid cellaggregates in affected tissues. Without being limited to any one theoryregarding B-cell mediated autoimmune disorder, it is believed that Bcells demonstrate a pathogenic effect in human autoimmune diseasesthrough a multitude of mechanistic pathways, including autoantibodyproduction, immune complex formation, dendritic and T-cell activation,cytokine synthesis, direct chemokine release, and providing a nidus forectopic neo-lymphogenesis. Each of these pathways may participate todifferent degrees in the pathology of autoimmune diseases.

As used herein, an “autoimmune disorder” can be an organ-specificdisease (i.e., the immune response is specifically directed against anorgan system such as the endocrine system, the hematopoietic system, theskin, the cardiopulmonary system, the gastrointestinal and liversystems, the renal system, the thyroid, the ears, the neuromuscularsystem, the central nervous system, etc.) or a systemic disease that canaffect multiple organ systems (for example, systemic lupus erythematosus(SLE), rheumatoid arthritis, polymyositis, etc.). Preferred suchdiseases include autoimmune rheumatologic disorders (such as, forexample, rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupussuch as SLE and lupus nephritis, polymyositis/dermatomyositis,cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriaticarthritis), autoimmune gastrointestinal and liver disorders (such as,for example, inflammatory bowel diseases (e.g., ulcerative colitis andCrohn's disease), autoimmune gastritis and pernicious anemia, autoimmunehepatitis, primary biliary cirrhosis, primary sclerosing cholangitis,and celiac disease), vasculitis (such as, for example, ANCA-negativevasculitis and ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and microscopic polyangiitis),autoimmune neurological disorders (such as, for example, multiplesclerosis (MS), opsoclonus myoclonus syndrome, myasthenia gravis,neuromyelitis optica, Parkinson's disease, Alzheimer's disease, andautoimmune polyneuropathies), renal disorders (such as, for example,glomerulonephritis, Goodpasture's syndrome, and Berger's disease),autoimmune dermatologic disorders (such as, for example, psoriasis,urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneouslupus erythematosus), hematologic disorders (such as, for example,thrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, autoimmune hearing diseases (such as, forexample, inner ear disease and hearing loss), Behcet's disease,Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders(such as, for example, diabetic-related autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), Addison's disease, andautoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, RA, IBD, includingCrohn's disease and ulcerative colitis, ANCA-associated vasculitis,lupus, MS, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis. Still more preferred are RA, IBD,lupus, and MS, and more preferred RA and IBD, and most preferred RA.

Specific examples of other autoimmune disorders as defined herein, whichin some cases encompass those listed above, include, but are not limitedto, arthritis (acute and chronic, rheumatoid arthritis includingjuvenile-onset rheumatoid arthritis and stages such as rheumatoidsynovitis, gout or gouty arthritis, acute immunological arthritis,chronic inflammatory arthritis, degenerative arthritis, type IIcollagen-induced arthritis, infectious arthritis, Lyme arthritis,proliferative arthritis, psoriatic arthritis, Still's disease, vertebralarthritis, osteoarthritis, arthritis chronica progrediente, arthritisdeformans, polyarthritis chronica primaria, reactive arthritis,menopausal arthritis, estrogen-depletion arthritis, and ankylosingspondylitis/rheumatoid spondylitis), autoimmune lymphoproliferativedisease, inflammatory hyperproliferative skin diseases, psoriasis suchas plaque psoriasis, gutatte psoriasis, pustular psoriasis, andpsoriasis of the nails, atopy including atopic diseases such as hayfever and Job's syndrome, dermatitis including contact dermatitis,chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis,allergic contact dermatitis, hives, dermatitis herpetiformis, nummulardermatitis, seborrheic dermatitis, non-specific dermatitis, primaryirritant contact dermatitis, and atopic dermatitis, x-linked hyper IgMsyndrome, allergic intraocular inflammatory diseases, urticaria such aschronic allergic urticaria and chronic idiopathic urticaria, includingchronic autoimmune urticaria, myositis, polymyositis/dermatomyositis,juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma(including systemic scleroderma), sclerosis such as systemic sclerosis,multiple sclerosis (MS) such as spino-optical MS, primary progressive MS(PPMS), and relapsing remitting MS (RRMS), progressive systemicsclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata,ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, gastrointestinal inflammation, colitis suchas ulcerative colitis, colitis ulcerosa, microscopic colitis,collagenous colitis, colitis polyposa, necrotizing enterocolitis, andtransmural colitis, and autoimmune inflammatory bowel disease), bowelinflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosingcholangitis, respiratory distress syndrome, including adult or acuterespiratory distress syndrome (ARDS), meningitis, inflammation of all orpart of the uvea, iritis, choroiditis, an autoimmune hematologicaldisorder, graft-versus-host disease, angioedema such as hereditaryangioedema, cranial nerve damage as in meningitis, herpes gestationis,pemphigoid gestationis, pruritis scroti, autoimmune premature ovarianfailure, sudden hearing loss due to an autoimmune condition,IgE-mediated diseases such as anaphylaxis and allergic and atopicrhinitis, encephalitis such as Rasmussen's encephalitis and limbicand/or brainstem encephalitis, uveitis, such as anterior uveitis, acuteanterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN(RPGN), proliferative nephritis, autoimmune polyglandular endocrinefailure, balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, food allergies, drug allergies, insect allergies, rareallergic disorders such as mastocytosis, allergic reaction, eczemaincluding allergic or atopic eczema, asteatotic eczema, dyshidroticeczema, and vesicular palmoplantar eczema, asthma such as asthmabronchiale, bronchial asthma, and auto-immune asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,immune reactions against foreign antigens such as fetal A-B-O bloodgroups during pregnancy, chronic pulmonary inflammatory disease,autoimmune myocarditis, leukocyte adhesion deficiency, lupus, includinglupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus,extra-renal lupus, discoid lupus and discoid lupus erythematosus,alopecia lupus, SLE, such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatric IDDM,adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes,idiopathic diabetes insipidus, diabetic retinopathy, diabeticnephropathy, diabetic colitis, diabetic large-artery disorder, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosisincluding lymphomatoid granulomatosis, agranulocytosis, vasculitides(including large-vessel vasculitis such as polymyalgia rheumatica andgiant-cell (Takayasu's) arteritis, medium-vessel vasculitis such asKawasaki's disease and polyarteritis nodosa/periarteritis nodosa,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as fibrinoid necrotizingvasculitis and systemic necrotizing vasculitis, ANCA-negativevasculitis, and ANCA-associated vasculitis such as Churg-Strausssyndrome (CSS), Wegener's granulomatosis, and microscopic polyangiitis),temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombspositive anemia, Diamond Blackfan anemia, hemolytic anemia or immunehemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia (anemia perniciosa), Addison's disease, pure red cellanemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia(s), cytopenias such as pancytopenia, leukopenia,diseases involving leukocyte diapedesis, CNS inflammatory disorders,Alzheimer's disease, Parkinson's disease, multiple organ injury syndromesuch as those secondary to septicemia, trauma or hemorrhage,antigen-antibody complex-mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, motoneuritis,allergic neuritis, Behçet's disease/syndrome, Castleman's syndrome,Goodpasture's syndrome, Reynaud's syndrome, Sjögren's syndrome,Stevens-Johnson syndrome, pemphigoid or pemphigus such as pemphigoidbullous, cicatricial (mucous membrane) pemphigoid, skin pemphigoid,pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceus,pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus,epidermolysis bullosa acquisita, ocular inflammation, preferablyallergic ocular inflammation such as allergic conjunctivis, linear IgAbullous disease, autoimmune-induced conjunctival inflammation,autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermalinjury due to an autoimmune condition, preeclampsia, an immune complexdisorder such as immune complex nephritis, antibody-mediated nephritis,neuroinflammatory disorders, polyneuropathies, chronic neuropathy suchas IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (asdeveloped by myocardial infarction patients, for example), includingthrombotic thrombocytopenic purpura (TTP), post-transfusion purpura(PTP), heparin-induced thrombocytopenia, and autoimmune orimmune-mediated thrombocytopenia including, for example, idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, Grave's eye disease (opthalmopathy orthyroid-associated opthalmopathy), polyglandular syndromes such asautoimmune polyglandular syndromes, for example, type I (orpolyglandular endocrinopathy syndromes), paraneoplastic syndromes,including neurologic paraneoplastic syndromes such as Lambert-Eatonmyasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-personsyndrome, encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, pneumonitis such as lymphoidinterstitial pneumonitis (LIP), bronchiolitis obliterans(non-transplant) vs. NSIP, Guillain-Barré syndrome, Berger's disease(IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis,acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis,transient acantholytic dermatosis, cirrhosis such as primary biliarycirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiacor Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia,amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronaryartery disease, autoimmune ear disease such as autoimmune inner eardisease (AIED), autoimmune hearing loss, polychondritis such asrefractory or relapsed or relapsing polychondritis, pulmonary alveolarproteinosis, keratitis such as Cogan's syndrome/nonsyphiliticinterstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosaceaautoimmune, zoster-associated pain, amyloidosis, a non-cancerouslymphocytosis, a primary lymphocytosis, which includes monoclonal B celllymphocytosis (e.g., benign monoclonal gammopathy and monoclonalgammopathy of undetermined significance, MGUS), peripheral neuropathy,paraneoplastic syndrome, channelopathies such as epilepsy, migraine,arrhythmia, muscular disorders, deafness, blindness, periodic paralysis,and channelopathies of the CNS, autism, inflammatory myopathy, focal orsegmental or focal segmental glomerulosclerosis (FSGS), endocrineopthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatologicaldisorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome,adrenalitis, gastric atrophy, presenile dementia, demyelinating diseasessuch as autoimmune demyelinating diseases and chronic inflammatorydemyelinating polyneuropathy, Dressler's syndrome, alopecia greata,alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon,esophageal dysmotility, sclerodactyl), and telangiectasia), male andfemale autoimmune infertility, e.g., due to anti-spermatozoanantibodies, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, parasitic diseases such as leishmaniasis, kypanosomiasis,schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan'ssyndrome, dengue, endocarditis, endomyocardial fibrosis, diffuseinterstitial pulmonary fibrosis, interstitial lung fibrosis, fibrosingmediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cysticfibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch'scyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV)infection, SCID, acquired immune deficiency syndrome (AIDS), echovirusinfection, sepsis (systemic inflammatory response syndrome (SIRS)),endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubellavirus infection, post-vaccination syndromes, congenital rubellainfection, Epstein-Barr virus infection, mumps, Evan's syndrome,autoimmune gonadal failure, Sydenham's chorea, post-streptococcalnephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,chorioiditis, giant-cell polymyalgia, chronic hypersensitivitypneumonitis, conjunctivitis, such as vernal catarrh,keratoconjunctivitis sicca, and epidemic keratoconjunctivitis,idiopathic nephritic syndrome, minimal change nephropathy, benignfamilial and ischemia-reperfusion injury, transplant organ reperfusion,retinal autoimmunity, joint inflammation, bronchitis, chronicobstructive airway/pulmonary disease, silicosis, aphthae, aphthousstomatitis, arteriosclerotic disorders (cerebral vascular insufficiency)such as arteriosclerotic encephalopathy and arterioscleroticretinopathy, aspermiogenese, autoimmune hemolysis, Boeck's disease,cryoglobulinemia, Dupuytren's contracture, endophthalmiaphacoanaphylactica, enteritis allergica, erythema nodosum leprosum,idiopathic facial paralysis, chronic fatigue syndrome, febrisrheumatica, Hamman-Rich's disease, sensoneural hearing loss,haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica (sympatheticophthalmitis), neonatal ophthalmitis, optic neuritis, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, lymphofollicular thymitis, vitiligo, toxic-shocksyndrome, food poisoning, conditions involving infiltration of T cells,leukocyte-adhesion deficiency, immune responses associated with acuteand delayed hypersensitivity mediated by cytokines and T-lymphocytes,diseases involving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, autoimmune polyendocrinopathies, oophoritis, primarymyxedema, autoimmune atrophic gastritis, rheumatic diseases, mixedconnective tissue disease, nephrotic syndrome, insulitis, polyendocrinefailure, autoimmune polyglandular syndromes, including polyglandularsyndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH),cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosaacquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome,primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acuteor chronic sinusitis, ethmoid, frontal, maxillary, or sphenoidsinusitis, allergic sinusitis, an eosinophil-related disorder such aseosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgiasyndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropicalpulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing eosinophils, anaphylaxis, spondyloarthropathies,seronegative spondyloarthritides, polyendocrine autoimmune disease,sclerosing cholangitis, sclera, episclera, chronic mucocutaneouscandidiasis, Bruton's syndrome, transient hypogammaglobulinemia ofinfancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome,angiectasis, autoimmune disorders associated with collagen disease,rheumatism such as chronic arthrorheumatism, lymphadenitis, reduction inblood pressure response, vascular dysfunction, tissue injury,cardiovascular ischemia, hyperalgesia, renal ischemia, cerebralischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,ischemic re-perfusion disorder, reperfusion injury of myocardial orother tissues, lymphomatous tracheobronchitis, inflammatory dermatoses,dermatoses with acute inflammatory components, multiple organ failure,bullous diseases, renal cortical necrosis, acute purulent meningitis orother central nervous system inflammatory disorders, ocular and orbitalinflammatory disorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

As used herein, “rheumatoid arthritis” or “RA” refers to a recognizeddisease state that may be diagnosed according to the 2000 revisedAmerican Rheumatoid Association criteria for the classification of RA,or any similar criteria, and includes active, early, and incipient RA,as defined below. Physiological indicators of RA include symmetric jointswelling, which is characteristic though not invariable in rheumatoidarthritis. Fusiform swelling of the proximal interphalangeal (PIP)joints of the hands as well as metacarpophalangeal (MCP), wrists,elbows, knees, ankles, and metatarsophalangeal (MTP) joints are commonlyaffected and swelling is easily detected. Pain on passive motion is themost sensitive test for joint inflammation, and inflammation andstructural deformity often limit the range of motion for the affectedjoint. Typical visible changes include ulnar deviation of the fingers atthe MCP joints, hyperextension, or hyperflexion of the MCP and PIPjoints, flexion contractures of the elbows, and subluxation of thecarpal bones and toes. The subject with RA may be resistant to DMARDs,in that the DMARDs are not effective or fully effective in treatingsymptoms. Further candidates for therapy according to this inventioninclude those who have experienced an inadequate response to previous orcurrent treatment with TNF inhibitors such as etanercept, infliximab,and/or adalimumab because of toxicity or inadequate efficacy (forexample, etanercept for 3 months at 25 mg twice a week or at least 4infusions of infliximab at 3 mg/kg).

A patient with “active rheumatoid arthritis” means a patient with activeand not latent symptoms of RA. Subjects with “early active rheumatoidarthritis” are those subjects with active RA diagnosed for at leasteight weeks but no longer than four years, according to the revised 1987ACR criteria for the classification of RA. Subjects with “earlyrheumatoid arthritis” are those subjects with RA diagnosed for at leasteight weeks but no longer than four years, according to the revised 1987ACR criteria for classification of RA. Early RA includes, for example,juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA(JRA).

Patients with “incipient RA” have early polyarthritis that does notfully meet ACR criteria for a diagnosis of RA, but is associated withthe presence of RA-specific prognostic biomarkers such as anti-CCP andshared epitope. They include patients with positive anti-CCP antibodieswho present with polyarthritis, but do not yet have a diagnosis of RA,and are at high risk for going on to develop bona fide ACR criteria RA(95% probability).

“Joint damage” is used in the broadest sense and refers to damage orpartial or complete destruction to any part of one or more joints,including the connective tissue and cartilage, where damage includesstructural and/or functional damage of any cause, and may or may notcause joint pain/arthalgia. It includes, without limitation, jointdamage associated with or resulting from inflammatory joint disease aswell as non-inflammatory joint disease. This damage may be caused by anycondition, such as an autoimmune disease, especially arthritis, and mostespecially RA. Exemplary such conditions include acute and chronicarthritis, RA including juvenile-onset RA, juvenile idiopathic arthritis(JIA), or juvenile RA (JRA), and stages such as rheumatoid synovitis,gout or gouty arthritis, acute immunological arthritis, chronicinflammatory arthritis, degenerative arthritis, type II collagen-inducedarthritis, infectious arthritis, septic arthritis, Lyme arthritis,proliferative arthritis, psoriatic arthritis, Still's disease, vertebralarthritis, osteoarthritis, arthritis chronica progrediente, arthritisdeformans, polyarthritis chronica primaria, reactive arthritis,menopausal arthritis, estrogen-depletion arthritis, and ankylosingspondylitis/rheumatoid spondylitis), rheumatic autoimmune disease otherthan RA, and significant systemic involvement secondary to RA (includingbut not limited to vasculitis, pulmonary fibrosis or Felty's syndrome).For purposes herein, joints are points of contact between elements of askeleton (of a vertebrate such as an animal) with the parts thatsurround and support it and include, but are not limited to, forexample, hips, joints between the vertebrae of the spine, joints betweenthe spine and pelvis (sacroiliac joints), joints where the tendons andligaments attach to bones, joints between the ribs and spine, shoulders,knees, feet, elbows, hands, fingers, ankles, and toes, but especiallyjoints in the hands and feet.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishing of any direct or indirect pathological consequences of thedisease, prevention of metastasis, decreasing of the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, the antibodies ofthe invention are used to delay development of a disease or disorder. Asubject is successfully “treated” for example, for an autoimmunedisorder if, after receiving a therapeutic amount of an antibody of theinvention according to the methods of the present invention, the subjectshows observable and/or measurable reduction in or absence of one ormore signs and symptoms of the particular disease.

In one preferred embodiment of successful treatment, the antibodyinduces a major clinical response in a subject with RA. For purposesherein, a “major clinical response” is defined as achieving an AmericanCollege of Rheumatology 70 response (ACR 70) for six consecutive months.ACR response scores are categorized as ACR 20, ACR 50, and ACR 70, withACR 70 being the highest level of sign and symptom control in thisevaluation system. ACR response scores measure improvement in RA diseaseactivity, including joint swelling and tenderness, pain, level ofdisability, and overall patient and physician assessment. An example ofa different type of antibody that induces a major clinical response asrecognized by the FDA and as defined herein is etanercept (ENBREL®).

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a medicament herein may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the medicament, e.g., antibody, toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of the drugin question, e.g., antibody, are outweighed by the therapeuticallybeneficial effects. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically but not necessarily,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopesof Lu), chemotherapeutic agents, and toxins such as small-moleculetoxins or enzymatically active toxins of bacterial, fungal, plant, oranimal origin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; CDP323, an oral alpha-4 integrin inhibitor;pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such aschlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such asthe enediyne antibiotics (e.g., calicheamicin, especially calicheamicingamma1I and calicheamicin omegall (see, e.g., Nicolaou et al., Angew.Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores); otherantibiotics such as aclacinomycin, actinomycin, authramycin, azaserine,bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin,chromomycin, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®),and deoxydoxorubicin), epirubicin, esorubicin, idarubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites such asmethotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine(XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acidanalogues such as denopterin, methotrexate, pteropterin, andtrimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine; anti-adrenals such asaminoglutethimide, mitotane, trilostane; and folic acid replenisher suchas frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK®polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane;rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g.,paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation ofpaclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil;6-thioguanine; mercaptopurine; platinum analogs such as cisplatin andcarboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16);ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin;leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and toremifene (FARESTON®);anti-progesterones; estrogen receptor down-regulators (ERDs); estrogenreceptor antagonists such as fulvestrant (FASLODEX®); agents thatfunction to suppress or shut down the ovaries, for example, leutinizinghormone-releasing hormone (LHRH) agonists such as leuprolide acetate(LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate andtripterelin; anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole,vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).In addition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinibditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-moleculeinhibitor also known as GW572016); and pharmaceutically acceptablesalts, acids, or derivatives of any of the above.

The term “cytokine” is a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof such cytokines are lymphokines, monokines, interleukins (ILs) such asIL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11,IL-12, and IL-15, including PROLEUKIN® rIL-2, a TNF such as TNF-α orTNF-β, and other polypeptide factors including leukocyte-inhibitoryfactor (LIF) and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native-sequence cytokines,including synthetically produced small-molecule entities andpharmaceutically acceptable derivatives and salts thereof. A “cytokineantagonist” is a molecule that inhibits or antagonizes such cytokines byany mechanism, including, for example, antibodies to the cytokine,antibodies to the cytokine receptor, and immunoadhesins.

For purposes herein, “tumor necrosis factor alpha” or “TNF-alpha” or“TNFα” refers to a human TNF-alpha molecule comprising the amino acidsequence as described in Pennica et al., Nature, 312:721 (1984) orAggarwal et al., J. Biol. Chem., 260:2345 (1985).

A “TNF antagonist” or “TNF inhibitor” is defined herein as a moleculethat decreases, blocks, inhibits, abrogates, or otherwise interfereswith TNFα activity in vitro, in situ, and/or preferably in vivo. Such anagent inhibits, to some extent, a biological function of TNF-alpha,generally through binding to TNF-alpha and neutralizing its activity. Asuitable TNF antagonist can also decrease block, abrogate, interfere,prevent and/or inhibit TNF RNA, DNA, or protein synthesis, TNFα release,TNFα receptor signaling, membrane TNFα cleavage, TNFα activity, and TNFαproduction and/or synthesis. Such TNF antagonists include, but are notlimited to, anti-TNFα antibodies, antigen-binding fragments thereof,specified mutants or domains thereof that bind specifically to TNFαthat, upon binding to TNFα, destroy or deplete cells expressing the TNFαin a mammal and/or interfere with one or more functions of those cells,a soluble TNF receptor (e.g., p55, p70 or p85) or fragment, fusionpolypeptides thereof, a small-molecule TNF antagonist, e.g., TNF bindingprotein I or II (TBP-I or TBP-II), nerelimonmab, CDP-571, infliximab(REMICADE®), etanercept (ENBREL®), adalimulab (HUMIRA™), CDP-571,CDP-870, afelimomab, lenercept, and the like), antigen-binding fragmentsthereof, and receptor molecules that bind specifically to TNFα,compounds that prevent and/or inhibit TNFα synthesis, TNFα release, orits action on target cells, such as thalidomide, tenidap,phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram), A2badenosine receptor agonists, and A2b adenosine receptor enhancers,compounds that prevent and/or inhibit TNFα receptor signaling, such asmitogen-activated protein (MAP) kinase inhibitors, compounds that blockand/or inhibit membrane TNFα cleavage, such as metalloproteinaseinhibitors, compounds that block and/or inhibit TNFα activity, such asangiotensin-converting enzyme (ACE) inhibitors (e.g., captopril), andcompounds that block and/or inhibit TNFα production and/or synthesis,such as MAP kinase inhibitors. The preferred antagonist comprises anantibody or an immunoadhesin. Examples of TNF antagonists specificallycontemplated herein are etanercept (ENBREL®), infliximab (REMICADE®),and adalimumab (HUMIRA™).

The term “hormone” refers to polypeptide hormones, which are generallysecreted by glandular organs with ducts. Included among the hormonesare, for example, growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; estradiol;hormone-replacement therapy; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, or testolactone;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);prolactin, placental lactogen, mouse gonadotropin-associated peptide,gonadotropin-releasing hormone; inhibin; activin; mullerian-inhibitingsubstance; and thrombopoietin. As used herein, the term hormone includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native-sequence hormone,including synthetically produced small-molecule entities andpharmaceutically acceptable derivatives and salts thereof.

The term “growth factor” refers to proteins that promote growth, andinclude, for example, hepatic growth factor; fibroblast growth factor;vascular endothelial growth factor; nerve growth factors such as NGF-β;platelet-derived growth factor; transforming growth factors (TGFs) suchas TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin(EPO); osteoinductive factors; interferons such as interferon-α, -β, and-γ; and colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF(G-CSF). As used herein, the term growth factor includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native-sequence growth factor, includingsynthetically produced small-molecule entities and pharmaceuticallyacceptable derivatives and salts thereof.

The term “integrin” refers to a receptor protein that allows cells bothto bind to and to respond to the extracellular matrix and is involved ina variety of cellular functions such as wound healing, celldifferentiation, homing of tumor cells, and apoptosis. They are part ofa large family of cell adhesion receptors that are involved incell-extracellular matrix and cell-cell interactions. Functionalintegrins consist of two transmembrane glycoprotein subunits, calledalpha and beta, that are non-covalently bound. The alpha subunits allshare some homology to each other, as do the beta subunits. Thereceptors always contain one alpha chain and one beta chain. Examplesinclude Alpha6beta1, Alpha3beta1, Alpha7beta1, LFA-1, etc. As usedherein, the term “integrin” includes proteins from natural sources orfrom recombinant cell culture and biologically active equivalents of thenative-sequence integrin, including synthetically producedsmall-molecule entities and pharmaceutically acceptable derivatives andsalts thereof.

An “integrin antagonist” is a molecule that inhibits or antagonizes suchintegrins by any mechanism, including, for example, antibodies to theintegrin. Examples of “integrin antagonists or antibodies” hereininclude an LFA-1 antibody, such as efalizumab (RAPTIVA®) commerciallyavailable from Genentech, or other CD11/11a and CD18 antibodies, or analpha 4 integrin antibody such as natalizumab (ANTEGREN®) available fromBiogen, or diazacyclic phenylalanine derivatives (WO 2003/89410),phenylalanine derivatives (WO 2003/70709, WO 2002/28830, WO 2002/16329and WO 2003/53926), phenylpropionic acid derivatives (WO 2003/10135),enamine derivatives (WO 2001/79173), propanoic acid derivatives (WO2000/37444), alkanoic acid derivatives (WO 2000/32575), substitutedphenyl derivatives (U.S. Pat. Nos. 6,677,339 and 6,348,463), aromaticamine derivatives (U.S. Pat. No. 6,369,229), ADAM disintegrin domainpolypeptides (US 2002/0042368), antibodies to alphavbeta3 integrin (EP633945), aza-bridged bicyclic amino acid derivatives (WO 2002/02556),etc.

“Corticosteroid” refers to any one of several synthetic or naturallyoccurring substances with the general chemical structure of steroidsthat mimic or augment the effects of the naturally occurringcorticosteroids. Examples of synthetic corticosteroids includeprednisone, prednisolone (including methylprednisolone, such asSOLU-MEDROL® methylprednisolone sodium succinate), dexamethasone ordexamethasone triamcinolone, hydrocortisone, and betamethasone. Thepreferred corticosteroids herein are prednisone, methylprednisolone,hydrocortisone, or dexamethasone.

The term “immunosuppressive agent” as used herein for adjunct therapyrefers to substances that act to suppress or mask the immune system ofthe mammal being treated herein. This would include substances thatsuppress cytokine production, down-regulate or suppress self-antigenexpression, or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,glucocorticoids such as cortisol or aldosterone, anti-inflammatoryagents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor,or a leukotriene receptor antagonist; purine antagonists such asazathioprine or mycophenolate mofetil (MMF); trocade (Ro32-355); aperipheral sigma receptor antagonist such as ISR-31747; alkylatingagents such as cyclophosphamide; bromocryptine; danazol; dapsone;glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat.No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHCfragments; cyclosporin A; steroids such as corticosteroids orglucocorticosteroids or glucocorticoid analogs, e.g., prednisone,methylprednisolone, including SOLU-MEDROL® methylprednisolone sodiumsuccinate, rimexolone, and dexamethasone; dihydrofolate reductaseinhibitors such as methotrexate (oral or subcutaneous); anti-malarialagents such as chloroquine and hydroxychloroquine; sulfasalazine;leflunomide; cytokine release inhibitors such as SB-210396 and SB-217969monoclonal antibodies and a MHC II antagonist such as ZD2315; a PG1receptor antagonist such as ZD4953; a VLA4 adhesion blocker such asZD7349; anti-cytokine or anti-cytokine receptor antibodies includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-TNF-alphaantibodies (infliximab (REMICAD®) or adalimumab), anti-TNF-alphaimmunoadhesin (etanercept), anti-TNF-beta antibodies, interleukin-1(IL-1) blockers such as recombinant HuIL-1Ra and IL-1B inhibitor,anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies;IL-2 fusion toxin; anti-L3T4 antibodies; leflunomide; heterologousanti-lymphocyte globulin; OPC-14597; NISV (immune response modifier); anessential fatty acid such as gammalinolenic acid or eicosapentaenoicacid; CD-4 blockers and pan-T antibodies, preferably anti-CD3 oranti-CD4/CD4a antibodies; co-stimulatory modifier (e.g., CTLA4-Fcfusion, also known as ABATACEP™); anti-interleukin-6 (IL-6) receptorantibodies and antagonists; anti-LFA-1 antibodies, including anti-CD 11aand anti-CD18 antibodies; soluble peptide containing a LFA-3-bindingdomain (WO 1990/08187); streptokinase; IL-10; transforming growthfactor-beta (TGF-beta); streptodornase; RNA or DNA from the host; FK506;RS-61443; enlimomab; CDP-855; PNP inhibitor; CH-3298; GW353430; 4162W94,chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al.,U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et al.,Science, 251: 430-432 (1991); WO 1990/11294; Janeway, Nature, 341:482-483 (1989); and WO 91/01133); BAFF antagonists such as BAFFantibodies and BR3 antibodies; zTNF4 antagonists (Mackay and Mackay,Trends Immunol., 23:113-5 (2002)); biologic agents that interfere with Tcell helper signals, such as anti-CD40 receptor or anti-CD40 ligand(CD154), including blocking antibodies to CD40-CD40 ligand (e.g., Durieet al., Science, 261: 1328-30 (1993); Mohan et al., J. Immunol., 154:1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265: 1225-7(1994)); and T-cell receptor antibodies (EP 340,109) such as T10B9. Somepreferred immunosuppressive agents herein include cyclophosphamide,chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

Examples of “disease-modifying anti-rheumatic drugs” or “DMARDs” includechloroquine, hydroxycloroquine, myocrisin, auranofin, sulfasalazine,methotrexate, leflunomide, etanercept, infliximab (plus oral andsubcutaneous methotrexate), azathioprine, D-penicilamine, gold salts(oral), gold salts (intramuscular), minocycline, cyclosporine includingcyclosporine A and topical cyclosporine, staphylococcal protein A(Goodyear and Silverman, J. Exp. Med., 197, (9), p 1125-39 (2003)),including salts and derivatives thereof, etc.

Examples of “non-steroidal anti-inflammatory drugs” or “NSAIDs” includeaspirin, acetylsalicylic acid, ibuprofen and ibuprofen retard,fenoprofen, piroxicam, flurbiprofen, naproxen, ketoprofen, naproxen,tenoxicam, benorylate, diclofenac, naproxen, nabumetone, indomethacin,ketoprofen, mefenamic acid, diclofenac, fenbufen, azapropazone,acemetacin, tiaprofenic acid, indomethacin, sulindac, tolmetin,phenylbutazone, diclofenac and diclofenac retard, cyclooxygenase (COX)-2inhibitors such as GR 253035, MK966, celecoxib (CELEBREX®;4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl),benzenesulfonamide and valdecoxib (BEXTRA®), and meloxicam (MOBIC®),including salts and derivatives thereof, etc. Preferably, they areaspirin, naproxen, ibuprofen, indomethacin, or tolmetin. Such NSAIDs areoptionally used with an analgesic such as codenine, tramadol, and/ordihydrocodinine or narcotic such as morphine.

A “B cell” is a lymphocyte that matures within the bone marrow, andincludes a naïve B cell, memory B cell, or effector B cell (plasmacells). The B cell herein may be a normal or non-malignant B cell.

The “CD20” antigen, or “CD20,” is an about 35-kDa, non-glycosylatedphosphoprotein found on the surface of greater than 90% of B cells fromperipheral blood or lymphoid organs. CD20 is present on both normal Bcells as well as malignant B cells, but is not expressed on stem cells.Other names for CD20 in the literature include “B-lymphocyte-restrictedantigen” and “Bp35”. The CD20 antigen is described in Clark et al.,Proc. Natl. Acad. Sci. (USA) 82:1766 (1985), for example. The preferredCD20 is non-human primate or human CD20, most preferably human CD20.

The “CD22” antigen, or “CD22,” also known as BL-CAM or Lyb8, is a type Iintegral membrane glycoprotein with molecular weight of about 130(reduced) to 140 kD (unreduced). It is expressed in both the cytoplasmand cell membrane of B-lymphocytes. CD22 antigen appears early in B-celllymphocyte differentiation at approximately the same stage as the CD19antigen. Unlike other B-cell markers, CD22 membrane expression islimited to the late differentiation stages comprised between mature Bcells (CD22+) and plasma cells (CD22−). The CD22 antigen is described,for example, in Wilson et al., J. Exp. Med. 173:137 (1991) and Wilson etal., J. Immunol. 150:5013 (1993).

An “antagonist to a B-cell surface marker” is a molecule that, uponbinding to a B-cell surface marker, destroys or depletes B cells in amammal and/or interferes with one or more B-cell functions, e.g. byreducing or preventing a humoral response elicited by the B cell. Theantagonist preferably is able to deplete B cells (i.e. reducecirculating B-cell levels) in a mammal treated therewith. Such depletionmay be achieved via various mechanisms such as ADCC and/or CDC,inhibition of B-cell proliferation and/or induction of B-cell death(e.g. via apoptosis). Antagonists included within the scope of thepresent invention include antibodies, synthetic or native-sequencepeptides, immunoadhesins, and small-molecule antagonists that bind to aB-cell surface marker, optionally conjugated with or fused to anothermolecule. The preferred antagonist comprises an antibody orimmunoadhesin. It includes BLyS antagonists such as immunoadhesins, andis preferably lumiliximab (anti-CD23), CD20, CD22, or BR3 antibody orBLyS immunoadhesin. The BLyS immunoadhesin preferably is selected fromthe group consisting of BR3 immunoadhesin comprising the extracellulardomain of BR3, TACI immunoadhesin comprising the extracellular domain ofTACI, and BCMA immunoadhesin comprising the extracellular domain ofBCMA. The most preferred BR3 immunoadhesin is hBR3-Fc of SEQ ID NO:2 ofWO 2005/00351 and US 2005/0095243. See also US 2005/0163775 and WO2006/068867. Another preferred BLyS antagonist is an anti-BLyS antibody,more preferably wherein the anti-BLyS antibody binds BLyS within aregion of BLyS comprising residues 162-275, or an anti-BR3 antibody,more preferably wherein the anti-BR3 antibody binds BR3 in a regioncomprising residues 23-38 of human BR3.

Examples of CD20 antibodies include: “C2B8,” which is now called“rituximab” (“RITUXAN®”) (U.S. Pat. No. 5,736,137) and fragments thereofthat retain the ability to bind CD20; the yttrium-[90]-labelled 2B8murine antibody designated “Y2B8” or “Ibritumomab Tiuxetan” (ZEVALIN®)commercially available from IDEC Pharmaceuticals, Inc. (U.S. Pat. No.5,736,137; 2B8 deposited with ATCC under accession no. HB11388 on Jun.22, 1993); murine IgG2a “B1,” also called “Tositumomab,” optionallylabelled with ¹³¹I to generate the “131I-B1” or “iodine I131 tositumomab” antibody (BEXXAR™) commercially available from Corixa (see,also, U.S. Pat. No. 5,595,721); murine monoclonal antibody “1F5” (Presset al. Blood 69(2):584-591 (1987) and variants thereof including“framework patched” or humanized 1F5 (WO 2003/002607, Leung, S.; ATCCdeposit HB-96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No.5,677,180); a humanized 2H7 (WO 2004/056312 (Lowman et al.) and as setforth below); HUMAX-CD20™ fully human, high-affinity antibody targetedat the CD20 molecule in the cell membrane of B-cells (Genmab, Denmark;see, for example, Glennie and van de Winkel, Drug Discovery Today 8:503-510 (2003) and Cragg et al., Blood 101: 1045-1052 (2003)); the humanmonoclonal antibodies set forth in WO 2004/035607 (Teeling et al.);AME-133™ antibodies (Applied Molecular Evolution); GA101 (GlycArt; US2005/0123546); A20 antibody or variants thereof such as chimeric orhumanized A20 antibody (cA20, hA20, respectively) (US 2003/0219433,Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-C1 orNU-B2 available from the International Leukocyte Typing Workshop(Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440,Oxford University Press (1987)). The preferred CD20 antibodies hereinare chimeric, humanized, or human CD20 antibodies, more preferablyrituximab, a humanized 2H7, chimeric or humanized A20 antibody(Immunomedics), and HUMAX-CD20™ human CD20 antibody (Genmab).

Purely for the purposes herein and unless indicated otherwise, a“humanized 2H7” refers to a humanized CD20 antibody, or anantigen-binding fragment thereof, wherein the antibody is effective todeplete primate B cells in vivo. The antibody includes those set forthin US 2006/0062787 and the figures thereof, and including thoseantibodies the sequences of which are provided in US 2006/0188495. Seealso US 2006/0034835 and US 2006/0024300. In a summary of variouspreferred embodiments of the invention, the V region of variants basedon 2H7 version 16 as disclosed in US 2006/0062787 will have the aminoacid sequences of v16 except at the positions of amino acidsubstitutions that are indicated in the table below. Unless otherwiseindicated, the 2H7 variants will have the same L chain as that of v16.

2H7 Heavy chain Light chain version (V_(H)) changes (V_(L)) changes Fcchanges 16 — A — — S298A, E333A, K334A B N100A M32L C N100A M32L S298A,E333A, K334A D D56A, S92A N100A E D56A, M32L, S92A S298A, E333A, K334AN100A F D56A, M32L, S92A S298A, E333A, K334A, N100A E356D, M358L G D56A,M32L, S92A S298A, K334A, K322A N100A H D56A, M32L, S92A S298A, E333A,K334A, N100A K326A I D56A, M32L, S92A S298A, E333A, K334A, N100A K326A,N434W J — — K334L

The preferred humanized 2H7 is an intact antibody or antibody fragmenthaving the sequence of version 16, or any of the versions shown above.

“BAFF antagonists” herein are any molecules that block the activity ofBAFF or BR3. They include immunoadhesins comprising a portion of BR3,TACI, or BCMA that binds BAFF, or variants thereof that bind BAFF. Inother embodiments, the BAFF antagonist is a BAFF antibody. A “BAFFantibody” is an antibody that binds BAFF, and preferably binds BAFFwithin a region of human BAFF comprising residues 162-275 of human BAFF.In another embodiment, the BAFF antagonist is BR3 antibody. A “BR3antibody” is an antibody that binds BR3, and is preferably one thatbinds BR3 within a region of human BR3 comprising residues 23-38 ofhuman BR3. The sequences of human BAFF and human BR3 are found, forexample, in US 2006/0062787. Other examples of BAFF-binding polypeptidesor BAFF antibodies can be found in, e.g., WO 2002/092620, WO2003/014294, Gordon et al., Biochemistry 42(20):5977-5983 (2003), Kelleyet al., J. Biol. Chem., 279(16):16727-16735 (2004), WO 1998/18921, WO2001/12812, WO 2000/68378 and WO 2000/40716.

“Chronic” administration refers to administration of the medicament(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH-buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low-molecular-weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

The term “mammal” refers to any animal classified as a mammal, includinghumans, domestic and farm animals, and zoo, sports, or pet animals, suchas dogs, horses, cats, cows, etc. Preferably, the mammal herein ishuman.

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications, usage, dosage, administration, contraindications, othermedicaments to be combined with the packaged product, and/or warningsconcerning the use of such medicaments, etc.

A “kit” is any manufacture (e.g., a package or container) comprising atleast one reagent, e.g., a medicament for treatment of an autoimmunedisease such as RA, lupus, MS, or IBD. The manufacture is preferablypromoted, distributed, or sold as a unit for performing the methods ofthe present invention.

A “target audience” is a group of people or an institution to whom or towhich a particular medicament is being promoted or intended to bepromoted, as by marketing or advertising, especially for particularuses, treatments, or indications, such as individual patients, patientpopulations, readers of newspapers, medical literature, and magazines,television or internet viewers, radio or internet listeners, physicians,drug companies, etc.

A “medicament” is an active drug to treat the disorder in question orits symptoms or side effects.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield.

Modes for Carrying Out the Invention

In one embodiment, the invention contemplates an isolated LTα antibodycomprising at least one CDR sequence selected from the group consistingof:

(a) a CDR-L1 sequence comprising amino acids A1-A11, wherein A1-A11 isKASQAVSSAVA (SEQ ID NO:1) or RASQAVSSAVA (SEQ ID NO:2), or comprisingamino acids A1-A17, wherein A1-A17 is KSSQSLLYSTNQKNFLA (SEQ ID NO:3) orKSSQSLLYSANQKNFLA (SEQ ID NO:4) or KSSQSLLYSTNQKNALA (SEQ ID NO:6),where N is any amino acid (chimeric 2C8 or humanized 2C8.v2/2C8.vX orchimeric 3F12/humanized 3F12.v5/humanized 3F12.v14, or 3F12 clone 14 or17, respectively);

(b) a CDR-L2 sequence comprising amino acids B1-B7, wherein B1-B7 isSASHRYT (SEQ ID NO:7) or WASTRDS (SEQ ID NO:8) (chimeric 2C8/humanized2C8.v2/humanized 2C8.vX or chimeric 3F12/humanized 3F12.v5/humanized3F12.v14, respectively);

(c) a CDR-L3 sequence comprising amino acids C1-C9, wherein C1-C9 isQQHYSTPWT (SEQ ID NO:9) or QENYSTPWT (SEQ ID NO:11) or QQYYSYPRT (SEQ IDNO:13) or QQYASYPRT (SEQ ID NO:14) or QQYYAYPRT (SEQ ID NO:15), where Nis any amino acid (chimeric 2C8/humanized 2C8.v2 or 2C8 clone G7 orchimeric 3F12/humanized 3F12.v5/humanized 3F12.v14 or 3F12 clone 20 or3F12 clone 21, respectively);

(d) a CDR-H1 sequence comprising amino acids D1-D10, wherein D1-D10 isGYTFTSYVIH (SEQ ID NO:16) or GYTFSSYWIE (SEQ ID NO:17) (chimeric2C8/humanized 2C8.v2/humanized 2C8.vX or chimeric 3F12/humanized3F12.v5/humanized 3F12.v14, respectively);

(e) a CDR-H2 sequence comprising amino acids E1-E17, wherein E1-E17 isYNNPYNDGTNYNEKFKG (SEQ ID NO:18) or EISPGSGSTNYNEEFKG (SEQ ID NO:19) orYNNPYNAGTNYNEKFKG (SEQ ID NO:101) or EINPGSGSTIYNEKFKG (SEQ ID NO:110),wherein N is any amino acid (chimeric 2C8/humanized 2C8.v2 or chimeric3F12/humanized 3F12.v5, or humanized 2C8.vX, or humanized 3F12.v14,respectively); and

(f) a CDR-H3 sequence comprising amino acids F1-F9, wherein F1-F9 isPTMLPWFAY (SEQ ID NO:20), or comprising amino acids F1-F5, wherein F1-F5is GYHGY (SEQ ID NO:21) or GYHGA (SEQ ID NO:22) (chimeric 2C8/humanized2C8.v2/humanized 2C8.vX or chimeric 3F12/humanized 3F12.v5/humanized3F12.v14 or 3F12 clone 12, respectively).

In one preferred embodiment, SEQ ID NO:3 is KSSQSLLYSTAQKNFLA (SEQ IDNO:5) (3F12 clone 15). In a further preferred embodiment, SEQ ID NO:11is QESYSTPWT (SEQ ID NO:10) (2C8 clone A8/humanized 2C8.vX) or QEVYSTPWT(SEQ ID NO:12) (2C8 clone H6).

In another preferred embodiment, the CDR-L1 sequence is SEQ ID NO:2 or 3or 4 or 6.

In another preferred aspect, the antibody comprises either (i) all ofthe CDR-L1 to CDR-L3 amino acid sequences of SEQ ID NOS:1 or 2 and 7 and9, or of SEQ ID NOS:1 or 2 and 7 or 8 and 11, or of SEQ ID NOS:3, 8, and13, or of SEQ ID NOS:4, 5, or 6, 8, and 13, or of SEQ ID NOS:3, 8, and14 or 15, or of SEQ ID NOS:4, 5, or 6, 8, and 14 or 15; or (ii) all ofthe CDR-H1 to CDR-H3 amino acid sequences of SEQ ID NOS:16, 18, and 20,or all of SEQ ID NOS:16, 101, and 20, or all of SEQ ID NOS:17, 19, and21 or 22, or all of SEQ ID NOS:17, 110, and 21.

In another aspect, the antibody comprises either all of SEQ ID NOS:1 or2 and 7 and 9, or all of SEQ ID NOS:16, 18, and 20, or all of SEQ IDNOS:16, 101, and 20. In an alternative embodiment, the antibodycomprises either all of SEQ ID NOS:3, 8, and 13, or all of SEQ IDNOS:17, 19, and 21 or 22. In an alternative embodiment, the antibodycomprises either all of SEQ ID NOS:4, 8, and 14, or all of SEQ IDNOS:17, 110, and 21. In other embodiments, the antibody comprises (i)all of the CDR-L1 to CDR-L3 amino acid sequences of SEQ ID NOS:1 or 2, 7and 9, or of SEQ ID NOS:1 or 2 and 7 or 8 and 11, of SEQ ID NOS:3, 8,and 13, or of SEQ ID NOS:4, 5, or 6, 8, and 13, or of SEQ ID NOS:3, 8,and 14 or 15, or of SEQ ID NOS:4, 5, or 6, 8, and 14 or 15; and (ii) allof the CDR-H1 to CDR-H3 amino acid sequences of SEQ ID NOS:16, 18, and20, or of SEQ ID NOS:16, 101, and 20, or of SEQ ID NOS:17, 19, and 21 or22, or of SEQ ID NOS:17, 110, and 21.

One preferred aspect is an anti-LTα antibody having a light-chainvariable domain comprising SEQ ID NO:23 or 24, or a heavy-chain variabledomain comprising SEQ ID NO:25 or 26, or having light-chain andheavy-chain variable domains comprising both SEQ ID NOS:23 and 25 orboth SEQ ID NOS:24 and 26. Further preferred is an anti-LTα antibodyhaving a light-chain variable domain comprising SEQ ID NO:27 or 28, or aheavy-chain variable domain comprising SEQ ID NO:29, 30, or 31, orhaving light-chain and heavy-chain variable domains comprising both SEQID NOS:27 and 29, or both SEQ ID NOS:27 and 30, or both SEQ ID NOS:27and 31, or both SEQ ID NOS:28 and 30, or both SEQ ID NOS:28 and 31. In astill further aspect, the invention provides an anti-LTα antibody havinga light-chain variable domain comprising SEQ ID NO:102, or a heavy-chainvariable domain comprising SEQ ID NO:103, or having light-chain andheavy-chain variable domains comprising both SEQ ID NOS:102 and 103. Ina still further aspect, the invention provides an anti-LTα antibodyhaving a light-chain variable domain comprising SEQ ID NO:108, or aheavy-chain variable domain comprising SEQ ID NO:109, or havinglight-chain and heavy-chain variable domains comprising both SEQ IDNOS:108 and 109.

Antibodies in other embodiments comprise a human κ subgroup 1 consensusframework sequence, and/or they comprise a heavy-chain human subgroupIII consensus framework sequence, wherein the framework sequencepreferably comprises a substitution at position 71, 73, and/or 78. Suchsubstitutions are preferably R71A, N73T, or N78A, or any combinationthereof, most preferably all three.

The antibodies of this invention preferably bind to LTα3 and block theinteraction of LTα3 with TNFRI and TNFRII. Preferably, they also bind toLTαβ complex and especially on the cell surface. Preferably, they alsoblock LTαβ function, and/or preferably contain an Fc region, and/ordecrease levels of inflammatory cytokines associated with RA in an invitro collagen-induced arthritis assay or an in vitro antibody-inducedarthritis assay.

Further preferred antibodies block the interaction of LTαβ with LTβ-Rand/or modulate LTαβ-expressing cells. In other preferred embodiments,the anti-LTα antibody herein substantially neutralizes at least oneactivity of at least one LTα protein. Preferably, the antibody hereintargets any cell expressing LTα, and preferably depletes LTα-positive or-secreting cells.

Preferred antibodies herein bind LTα with an affinity of at least about10⁻¹² M, more preferably at least about 10⁻¹³ M. The antibodies alsopreferably are of the IgG isotype, such as IgG1, IgG2a, IgG2b, or IgG3,more preferably human IgG, and most preferably IgG1 or IgG2a.

Another preferred antibody has a monovalent affinity to human LTα thatis about the same as or greater than the monovalent affinity to humanLTα of a murine antibody produced by a hybridoma cell line depositedunder American Type Culture Collection Accession Number PTA-7538(hybridoma murine Lymphotoxin alpha2 beta1 s5H3.2.2).

As is well established in the art, binding affinity of a ligand to itsreceptor can be determined using any of a variety of assays, andexpressed in terms of a variety of quantitative values. Accordingly, inone embodiment, the binding affinity is expressed as Kd values andreflects intrinsic binding affinity (e.g., with minimized avidityeffects). Generally and preferably, binding affinity is measured invitro, whether in a cell-free or cell-associated setting. Folddifference in binding affinity can be quantified in terms of the ratioof the monovalent binding affinity value of a humanized antibody (e.g.,in Fab form) and the monovalent binding affinity value of areference/comparator antibody (e.g., in Fab form) (e.g., a murineantibody having donor CDR sequences), wherein the binding affinityvalues are determined under similar assay conditions.

Thus, in one embodiment, the fold difference in binding affinity isdetermined as the ratio of the Kd values of the humanized antibody inFab form and said reference/comparator Fab antibody. For example, in oneembodiment, if an antibody of the invention (A) has an affinity that is“three-fold lower” than the affinity of a reference antibody (M), thenif the Kd value for A is 3x, the Kd value of M would be 1x, and theratio of Kd of A to Kd of M would be 3:1. Conversely, in one embodiment,if an antibody of the invention (C) has an affinity that is “three-foldgreater” than the affinity of a reference antibody (R), then if the Kdvalue for C is 1x, the Kd value of R would be 3x, and the ratio of Kd ofC to Kd of R would be 1:3. Any assays known in the art, including thosedescribed herein, can be used to obtain binding affinity measurements,including, for example, an optical biosensor that uses SPR (BIACORE®technology), RIA, and ELISA. Preferably, the measurement is by opticalbiosensor or radioimmunoassay.

The antibodies herein are preferably chimeric or humanized, morepreferably humanized, and still more preferably antibodies wherein atleast a portion of their framework sequence is a human consensusframework sequence.

Another embodiment herein is an anti-idiotype antibody that specificallybinds the antibody herein.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives, e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Rockville, Md. USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as RIA or ELISA.

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal, e.g, by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-SEPHAROSE™medium) or ion-exchange chromatography, hydroxylapatite chromatography,gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high-affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting humanheavy-chain and light-chain constant-domain (C_(H) and C_(L)) sequencesfor the homologous murine sequences (U.S. Pat. No. 4,816,567 andMorrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or byfusing the immunoglobulin coding sequence with all or part of the codingsequence for a non-immunoglobulin polypeptide (heterologouspolypeptide). The non-immunoglobulin polypeptide sequences cansubstitute for the constant domains of an antibody, or they aresubstituted for the variable domains of one antigen-combining site of anantibody to create a chimeric bivalent antibody comprising oneantigen-combining site having specificity for an antigen and anotherantigen-combining site having specificity for a different antigen.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source that is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.Nature 321:522-525 (1986); Riechmann et al. Nature 332:323-327 (1988);Verhoeyen et al. Science 239:1534-1536 (1988)), by substituting CDRsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567) wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al., J. Immunol.151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al. J. Immunol., 151:2623 (1993).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available that illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding.

The humanized antibody may be an antibody fragment, such as a Fab, whichis optionally conjugated with one or more cytotoxic agent(s) in order togenerate an immunoconjugate. Alternatively, the humanized antibody maybe a full-length antibody, such as a full-length IgG1 antibody.

Human Antibodies and Phage Display Methodology

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); and U.S. Pat. No. 5,545,807; and WO1997/17852.

Alternatively, phage-display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the LTα cell. Phage display can be performed in avariety of formats, reviewed in, e.g, Johnson and Chiswell, CurrentOpinion in Structural Biology 3:564-571 (1993). Several sources ofV-gene segments can be used for phage display. Clackson et al., Nature,352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of immunized mice. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al., J. Mol. Biol. 222:581-597(1991) or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S.Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al, Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See, e.g., WO 1993/16185; U.S. Pat. Nos.5,571,894; and 5,587,458. Fv and sFv are the only species with intactcombining sites that are devoid of constant regions; thus, they aresuitable for reduced nonspecific binding during in vivo use. sFv fusionproteins may be constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an sFv. The antibodyfragment may also be a “linear antibody”, e.g., as described in U.S.Pat. No. 5,641,870 for example. Such linear antibody fragments may bemonospecific or bispecific.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the LTα protein. Other such antibodiesmay combine a LTα binding site with a binding site for another protein.Alternatively, an anti-LTα arm may be combined with an arm that binds toa triggering molecule on a leukocyte such as a T-cell receptor molecule(e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16), or NKG2D or other NK-cell-activating ligand,so as to focus and localize cellular defense mechanisms to theLTβ-expressing cell. Bispecific antibodies may also be used to localizecytotoxic agents to cells that express LTα. These antibodies possess aLTα-binding arm and an arm that binds the cytotoxic agent (e.g.,saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull-length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies).

WO 1996/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibodyand U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRIantibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO1998/02463. U.S. Pat. No. 5,821,337 teaches a bispecificanti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy-chain/light-chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 1993/08829, and in Traunecker et al.,EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant-domain sequences. Preferably, thefusion is with an Ig heavy-chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light-chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy-chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant effect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulinheavy-chain/light-chain pair (providing a second binding specificity) inthe other arm. It was found that this asymmetric structure facilitatesthe separation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 1994/04690.For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 121:210 (1986).

In another approach (U.S. Pat. No. 5,731,168), the interface between apair of antibody molecules can be engineered to maximize the percentageof heterodimers that are recovered from recombinant cell culture. Thepreferred interface comprises at least a part of the C_(H)3 domain. Inthis method, one or more small amino acid side chains from the interfaceof the first antibody molecule are replaced with larger side chains(e.g., tyrosine or tryptophan). Compensatory “cavities” of identical orsimilar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chainswith smaller ones (e.g., alanine or threonine). This provides amechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO1991/00360, WO 1992/20373, and EP 03089). Heteroconjugate antibodies maybe made using any convenient cross-linking methods. Suitablecross-linking agents are well known in the art, and are disclosed, forexample, in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describes a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describesthe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Holliger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a V_(H)connected to a V_(L) by a linker that is too short to allow pairingbetween the two domains on the same chain. Accordingly, the V_(H) andV_(L) domains of one fragment are forced to pair with the complementaryV_(L) and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen-binding sites (e.g., tetravalent antibodies),which can be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or moreantigen-binding sites. The preferred dimerization domain comprises (orconsists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. The preferred multivalentantibody herein comprises (or consists of) three to about eight, butpreferably four, antigen-binding sites. The multivalent antibodycomprises at least one polypeptide chain (and preferably two polypeptidechains), wherein the polypeptide chain(s) comprise two or more variabledomains. For instance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable-domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light-chain variable-domainpolypeptides. The light-chain variable-domain polypeptides contemplatedherein comprise a light-chain variable-domain and, optionally, furthercomprise a CL domain.

Vectors, Host Cells, and Recombinant Methods

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the recombinant monoclonalantibodies, immunoadhesins, and other polypeptide antagonists describedherein are prokaryote, yeast, or higher eukaryotic cells. Suitableprokaryotes for this purpose include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

Full-length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector functions are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full-length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos.5,648,237; 5,789,199; and 5,840,523, which describetranslation-initiation region (TIR) and signal sequences for optimizingexpression and secretion. After expression, the antibody is isolatedfrom the E. coli cell paste in a soluble fraction and can be purifiedthrough, e.g., a protein A or G column depending on the isotype. Finalpurification can be carried out similar to the process for purifyingantibody expressed e.g, in CHO cells. For general monoclonal antibodyproduction, see also U.S. Pat. No. 7,011,974.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding, such as LTα-antibody-encoding, vectors. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of, e.g., glycosylatedLTα-binding antibody are derived from multicellular organisms. Examplesof invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (e.g., 293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, e.g., ATCC CCL 10); Chinese hamster ovarycells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)), including, but not limited to CHO K1, CHO pro3⁻, CHO DG44, CHODUXB11, Lec13, B-Ly1, and CHO DP12 cells, preferably a CHO DUX (DHFR-)or subclone thereof (herein called “CHO DUX”); C127 cells, mouse Lcells; Ltk⁻ cells; mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse myeloma cells;NS0; hybridoma cells such as mouse hybridoma cells; COS cells; mousemammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., AnnalsN.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a humanhepatoma line (Hep G2).

Host cells are transformed with expression or cloning vectors forproduction of the LTβ-binding antibody herein, and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 1990/03430; WO 1987/00195; or US Re. 30,985may be used as culture media for the host cells. Any of these media maybe supplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

In one particular aspect, a suitable medium contains a basal mediumcomponent such as a DMEM/HAM F-12 based formulation (for composition ofDMEM and HAM F12 media and especially serum-free media, see culturemedia formulations in the American Type Culture Collection Catalogue ofCell Lines and Hybridomas, Sixth Edition, 1988, pages 346-349) (theformulations of medium as described in U.S. Pat. No. 5,122,469 may beappropriate) with suitably modified, if necessary, concentrations ofsome components such as amino acids, salts, sugar, and vitamins, andoptionally containing glycine, hypoxanthine, and thymidine; recombinanthuman insulin, hydrolyzed peptone, such as PROTEASE PEPTONE 2 and 3™,PRIMATONE HS™ or PRIMATONE RL™ (Difco, USA; Sheffield, England), or theequivalent; a cell-protective agent, such as PLURONIC F68™ or theequivalent PLURONIC™ polyol; GENTAMYCIN™ antibiotic; and trace elements.Preferably the cell culture media is serum free.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least about 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small-scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about one liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al., JBio Chem 274:19601-19605 (1999); U.S. Pat. Nos. 6,083,715 and 6,027,888;Bothmann and Pluckthun, J. Biol. Chem. 275:17100-17105 (2000); Ramm andPluckthun, J. Biol. Chem. 275:17106-17113 (2000); and Arie et al., Mol.Microbiol. 39:199-210 (2001).

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, U.S. Pat. Nos.5,264,365; 5,508,192; and Hara et al., Microbial Drug Resistance,2:63-72 (1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system herein.

Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an AMICON™ or MILLIPORE PELLICON™ ultrafiltrationunit. A protease inhibitor such as phenylmethylsulphonylfluoride (PMSF)may be included in any of the foregoing steps to inhibit proteolysis,and antibiotics may be included to prevent the growth of adventitiouscontaminants.

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: hydroxylapatite chromatography,chromatography on heparin SEPHAROSE™, gel electrophoresis, dialysis,fractionation on immunoaffinity columns, ethanol precipitation,reverse-phase HPLC, chromatography on silica, chromatography on ananion- or cation-exchange resin (such as DEAE or a polyaspartic acidcolumn), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, andgel filtration using, for example, SEPHADEX™ G-75 resin. Affinitychromatography is a preferred purification technique. Foranalytical-scale purification, smaller volumes are passed throughcolumns and used; for preparative- or commercial-scale purification toproduce quantities of antibody useful in therapeutic applications,larger volumes are employed. The skilled artisan will understand whichscale should be used for which application. Preferably, preparativescale is employed for this invention.

The suitability of protein A as an affinity ligand depends on thespecies and isotype of any immunoglobulin Fc domain that is present inthe antibody. Protein A can be used to purify antibodies that are basedon human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such ascontrolled-pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. Where the antibody comprises a C_(H)3 domain, the BAKERBONDABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.

For Protein A chromatography, the solid phase to which Protein A isimmobilized is preferably a column comprising a glass or silica surface,more preferably a controlled pore glass column or a silicic acid column.In some applications, the column has been coated with a reagent, such asglycerol, to prevent nonspecific adherence of contaminants. The solidphase is then washed to remove contaminants non-specifically bound tothe solid phase. Finally, the antibody of interest is recovered from thesolid phase by elution.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic-interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0 to 0.25M salt).

Generating Variant Antibodies Exhibiting Reduced or Absence of HAMAResponse

Reduction or elimination of a HAMA response is a significant aspect ofclinical development of suitable therapeutic agents. See, e.g.,Khaxzaeli et al., J. Natl. Cancer Inst., 80:937 (1988); Jaffers et al.,Transplantation, 41:572 (1986); Shawler et al., J. Immunol., 135:1530(1985); Sears et al., J. Biol. Response Mod, 3:138 (1984); Miller etal., Blood, 62:988 (1983); Hakimi et al., J. Immunol., 147:1352 (1991);Reichmann et al., Nature, 332:323 (1988); and Junghans et al., CancerRes, 50:1495 (1990). In some embodiments herein, the invention providesantibodies that are humanized such that HAMA response is reduced oreliminated. Variants of these antibodies can further be obtained usingroutine methods known in the art, some of which are further describedbelow.

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or CDR sequence(s). A selected framework sequence to whicha starting CDR sequence is linked is referred to herein as an acceptorhuman framework. While the acceptor human frameworks may be from, orderived from, a human immunoglobulin (the VL and/or VH regions thereof),preferably the acceptor human frameworks are from, or derived from, ahuman consensus framework sequence, as such frameworks have beendemonstrated to have minimal, or no, immunogenicity in human patients.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence based on its homology tothe donor framework sequence by aligning the donor framework sequencewith various human framework sequences in a collection of humanframework sequences and selecting the most homologous framework sequenceas the acceptor.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup III and/or VL kappa subgroup I consensusframework sequences.

Thus, the VH acceptor human framework may comprise one, two, three, orall of the following framework sequences:

FR1 comprising EVQLVESGGGLVQPGGSLRLSCAAS, (SEQ ID NO: 46) FR2 comprisingWVRQAPGKGLEWVG, (SEQ ID NO: 47) FR3 comprising FR3 comprisesRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR, (SEQ ID NO: 48) orRATFSADNSKNTAYLQMNSLRAEDTAVYYCAD, (SEQ ID NO: 50) and FR4 comprisingWGQGTLVTVSS. (SEQ ID NO: 49)

The VL acceptor human framework may comprise one, two, three, or all ofthe following framework sequences:

FR1 comprising DIQMTQSPSSLSASVGDRVTITC, (SEQ ID NO: 51) FR2 comprisingWYQQKPGKAPKLQIY, (SEQ ID NO: 52) or WYQQKPGKAPKLLIY, (SEQ ID NO: 55)FR3 comprising GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, (SEQ ID NO: 53) andFR4 comprising FGQGTKVEIKR. (SEQ ID NO: 54)

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that be from a human immunoglobulin or ahuman consensus framework, the present invention contemplates that theacceptor sequence may comprise pre-existing amino acid substitutionsrelative to the human immunoglobulin sequence or human consensusframework sequence. These pre-existing substitutions are preferablyminimal, usually only four, three, two, or one amino acid differencerelative to the human immunoglobulin sequence or consensus frameworksequence.

CDR residues of the non-human antibody are incorporated into the VLand/or VH acceptor human frameworks. For example, one may incorporateresidues corresponding to the Kabat CDR residues, the Chothiahypervariable loop residues, the AbM residues, and/or the contactresidues. Optionally, the extended CDR residues as follows areincorporated: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35 (H1), 50-65or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3).

“Incorporation” of CDR residues can be achieved in various ways, e.g.,nucleic acid encoding the desired amino acid sequence can be generatedby mutating nucleic acid encoding the mouse variable domain sequence sothat the framework residues thereof are changed to acceptor humanframework residues, or by mutating nucleic acid encoding the humanvariable domain sequence so that the CDR residues are changed tonon-human residues, or by synthesizing nucleic acid encoding the desiredsequence, etc.

CDR-grafted variants may be generated by Kunkel mutagenesis of nucleicacid encoding the human acceptor sequences, using a separateoligonucleotide for each CDR. Kunkel et al., Methods Enzymol.154:367-382 (1987). Appropriate changes can be introduced within theframework and/or CDR, using routine techniques, to correct andre-establish proper CDR-antigen interactions.

Phage(mid) display (also referred to herein as phage display in somecontexts) can be used as a convenient and fast method for generating andscreening many different potential variant antibodies in a librarygenerated by sequence randomization. However, other methods for makingand screening altered antibodies are available to the skilled person.

Phage(mid) display technology has provided a powerful tool forgenerating and selecting novel proteins that bind to a ligand, such asan antigen. Using the techniques of phage(mid) display allows thegeneration of large libraries of protein variants that can be rapidlysorted for those sequences that bind to a target molecule with highaffinity. Nucleic acids encoding variant polypeptides are generallyfused to a nucleic acid sequence encoding a viral coat protein, such asthe gene III protein or the gene VIII protein. Monovalent phagemiddisplay systems where the nucleic acid sequence encoding the protein orpolypeptide is fused to a nucleic acid sequence encoding a portion ofthe gene III protein have been developed. (Bass, Proteins, 8:309 (1990);Lowman and Wells, Methods. A Companion to Methods in Enzymology, 3:205(1991)). In a monovalent phagemid display system, the gene fusion isexpressed at low levels and wild-type gene III proteins are alsoexpressed so that infectivity of the particles is retained. Methods ofgenerating peptide libraries and screening those libraries have beendisclosed in many patents (e.g., U.S. Pat. Nos. 5,723,286; 5,432,018;5,580,717; 5,427,908; and 5,498,530).

Libraries of antibodies have been prepared in a number of ways includingby altering a single gene by inserting random DNA sequences or cloning afamily of related genes. Methods for displaying antibodies orantigen-binding fragments using phage(mid) display are described in U.S.Pat. Nos. 5,750,373; 5,733,743; 5,837,242; 5,969,108; 6,172,197;5,580,717; and 5,658,727. The library is then screened for expression ofantibodies or antigen-binding proteins with the desired characteristics.

The sequence of oligonucleotides includes one or more of the designedcodon sets for the CDR residues to be altered. A codon set is a set ofdifferent nucleotide triplet sequences used to encode desired variantamino acids. Codon sets can be represented using symbols to designateparticular nucleotides or equimolar mixtures of nucleotides as shownbelow according to the IUB code.

IUB CODES G Guanine A Adenine T Thymine C Cytosine R (A or G) Y (C or T)M (A or C) K (G or T) S (C or G) W (A or T) H (A or C or T) B (C or G orT) V (A or C or G) D (A or G or T) H N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; Vcan be A or G or C; and K can be G or T. This codon set can present 18different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr,Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Oligonucleotide or primer sets can be synthesized using standardmethods. A set of oligonucleotides can be synthesized, for example, bysolid-phase synthesis, containing sequences that represent all possiblecombinations of nucleotide triplets provided by the codon set and thatwill encode the desired group of amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art. Such sets of nucleotides havingcertain codon sets can be synthesized using commercial nucleic acidsynthesizers (available from, for example, Applied Biosystems, FosterCity, Calif.), or can be obtained commercially (for example, from LifeTechnologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used herein, have sequences that allow forhybridization to a variable-domain nucleic acid template and also caninclude restriction enzyme sites for cloning purposes.

In one method, nucleic acid sequences encoding variant amino acids canbe created by oligonucleotide-mediated mutagenesis. This technique iswell known in the art as described by Zoller et al. Nucleic Acids Res.10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variantamino acids are created by hybridizing an oligonucleotide set encodingthe desired codon sets to a DNA template, where the template is thesingle-stranded form of the plasmid containing a variable-region nucleicacid template sequence. After hybridization, DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will contain thecodon sets as provided by the oligonucleotide set.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation(s). This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that in Crea et al., Proc. Nat'l.Acad. Sci. USA, 75:5765 (1978).

The DNA template is generated by those vectors that are derived frombacteriophage M13 vectors (the commercially available M13 mp 18 and M13mp 19 vectors are suitable), or that contain a single-stranded phageorigin of replication as described by Viera et al., Meth. Enzymol.,153:3 (1987). Thus, the DNA to be mutated can be inserted into one ofthese vectors to generate single-stranded template. Production of thesingle-stranded template is described in sections 4.21-4.41 of Sambrooket al., supra.

To alter the native DNA sequence, the oligonucleotide is hybridized tothe single stranded template under suitable hybridization conditions. ADNA-polymerizing enzyme, for example, T7 DNA polymerase or the Klenowfragment of DNA polymerase I, is then added to synthesize thecomplementary strand of the template using the oligonucleotide as aprimer for synthesis. A heteroduplex molecule is thus formed such thatone strand of DNA encodes the mutated form of gene 1, and the otherstrand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitablehost cell, usually a prokaryote such as E. coli JM101. After growing thecells, they are plated onto agarose plates and screened using theoligonucleotide primer radiolabeled with a ³²P phosphate to identify thebacterial colonies that contain the mutated DNA.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTT), is combined with a modifiedthiodeoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham). This mixture is added to the template-oligonucleotidecomplex. Upon addition of DNA polymerase to this mixture, a strand ofDNA identical to the template except for the mutated bases is generated.In addition, this new strand of DNA will contain dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonucleasedigestion. After the template strand of the double-stranded heteroduplexis nicked with an appropriate restriction enzyme, the template strandcan be digested with ExoIII nuclease or another appropriate nucleasepast the region that contains the site(s) to be mutagenized. Thereaction is then stopped to leave a molecule that is only partiallysingle-stranded. A complete double-stranded DNA homoduplex is thenformed using DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplexmolecule can then be transformed into a suitable host cell.

As indicated previously, the sequence of the oligonucleotide set is ofsufficient length to hybridize to the template nucleic acid and mayalso, but does not necessarily, contain restriction sites. The DNAtemplate can be generated by those vectors that are either derived frombacteriophage M13 vectors or vectors that contain a single-strandedphage origin of replication as described by Viera et al. Meth. Enzymol.,153:3 (1987). Thus, the DNA that is to be mutated must be inserted intoone of these vectors in order to generate single-stranded template.Production of the single-stranded template is described in sections4.21-4.41 of Sambrook et al., supra.

In another method, a library can be generated by providing upstream anddownstream oligonucleotide sets, each set having a plurality ofoligonucleotides with different sequences. These sequences areestablished by the codon sets provided within the sequence of theoligonucleotides. The upstream and downstream oligonucleotide sets,along with a variable-domain template nucleic acid sequence, can be usedin a polymerase chain reaction (PCR) to generate a “library” of PCRproducts. The PCR products can be referred to as “nucleic acidcassettes”, as they can be fused with other related or unrelated nucleicacid sequences, for example, viral coat proteins and dimerizationdomains, using established molecular biology techniques.

The sequence of the PCR primers includes one or more of the designedcodon sets for the solvent-accessible and highly diverse positions in aCDR. As described above, a codon set is a set of different nucleotidetriplet sequences used to encode desired variant amino acids.

Antibody selectants that meet the desired criteria, as selected throughappropriate screening/selection steps, can be isolated and cloned usingstandard recombinant techniques.

Activity Assays

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

The purified immunoglobulins can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino-acidanalysis, non-denaturing size-exclusion high-pressure liquidchromatography (HPLC), mass spectrometry, ion-exchange chromatography,and papain digestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies herein are tested for their antigen-binding activity. Theantigen-binding assays known in the art and useful herein include,without limitation, any direct or competitive-binding assays usingtechniques such as Western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, fluorescent immunoassays, and protein Aimmunoassays.

In one embodiment, the present invention contemplates an alteredantibody that possesses some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important, yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include PBMCs and NK cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. To assess complement activation,a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol.Methods 202:163 (1996), may be performed. FcRn binding and in vivoclearance/half life determinations can also be performed using methodsknown in the art.

Antibody Variants

In some embodiments, amino-acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced into the subject antibody amino acid sequence at the timethat the sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called“alanine-scanning mutagenesis” as described by Cunningham and Wells,Science, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g., for ADEPT) or a polypeptide that increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the CDRs, but FR alterations are alsocontemplated. Conservative substitutions are shown in Table A under theheading of “preferred substitutions.” If such substitutions result in achange in biological activity, then more substantial changes,denominated “exemplary substitutions” in Table A, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE A Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (as described, for example, in A. L. Lehninger, Biochemistry,second ed., pp. 73-75, Worth Publishers, New York (1975)):

-   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F),    Trp (W), Met (M)-   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),    Asn (N), Gln (O)-   (3) acidic: Asp (D), Glu (E)-   (4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

One type of substitutional variant involves substituting one or more CDRresidues of a parent antibody (e.g., a humanized or human antibody).Generally, the resulting variant(s) selected for further developmentwill have improved biological properties relative to the parent antibodyfrom which they are generated. A convenient way for generating suchsubstitutional variants involves affinity maturation using phagedisplay. Briefly, several CDR sites (e.g., 6-7 sites) are mutated togenerate all possible amino acid substitutions at each site. Theantibodies thus generated are displayed from filamentous phage particlesas fusions to the gene III product of M13 packaged within each particle.The phage-displayed variants are then screened for their biologicalactivity (e.g., binding affinity) as herein disclosed. For location ofcandidate CDR sites for modification, alanine-scanning mutagenesis canbe performed to identify CDR residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according tothe techniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

The antibodies of the present invention preferably have thenative-sequence Fc region. However, it may be desirable to introduce oneor more amino acid modifications into an Fc region thereof, generating aFc-region variant. The Fc-region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g., a substitution) at one or more aminoacid positions including that of a hinge cysteine. These antibodieswould nonetheless retain substantially the same characteristics requiredfor therapeutic utility as compared to their wild-type counterpart. Suchamino acid substitutions may, e.g., improve or reduce other Fc functionor further improve the same Fc function, increase antigen-bindingaffinity, increase stability, alter glycosylation, or include allotypicvariants. The antibodies may further comprise one or more amino acidsubstitutions in the Fc region that result in the antibody exhibitingone or more of the properties selected from increased FcγR binding,increased ADCC, increased CDC, decreased CDC, increased ADCC and CDCfunction, increased ADCC but decreased CDC function (e.g., to minimizeinfusion reaction), increased FcRn binding, and increased serum halflife, as compared to the same antibodies that have the wild-type Fcregion. These activities can be measured by the methods describedherein. For example, see WO 1999/51642. See also Duncan and Winter,Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No.5,624,821; and WO 1994/29351 concerning other examples of Fcregionvariants.

For additional amino acid alterations that improve Fc function, see,e.g., U.S. Pat. No. 6,737,056. Any of the antibodies of the presentinvention may further comprise at least one amino acid substitution inthe Fc region that decreases CDC activity, for example, comprising atleast the substitution K322A. See U.S. Pat. No. 6,528,624 (Idusogie etal.).

In another preferred embodiment, the antibody has amino acidsubstitutions at any one or any combination of positions that are 268D,or 298A, or 326D, or 333A, or 334A, or 298A together with 333A, or 298Atogether with 334A, or 239D together with 332E, or 239D together with298A and 332E, or 239D together with 268D and 298A and 332E, or 239Dtogether with 268D and 298A and 326A and 332A, or 239D together with268D and 298A and 326A and 332E, or 239D together with 268D and 283L and298A and 332E, or 239D together with 268D and 283L and 298A and 326A and332E, or 239D together with 330L and 332E and 272Y and 254T and 256E, or250Q together with 428L, or 265A, or 297A, wherein the 265A substitutionis in the absence of 297A and the 297A substitution is in the absence of265A. The letter after the number in each of these designationsrepresents the changed amino acid at that position.

Mutations that improve ADCC and CDC include substitutions at one tothree positions of the Fc region, including positions 298, 333, and/or334 of the Fc region (Eu numbering of residues), especially S298Atogether with E333A and K334A (S298A/E333A/K334A, or synonymously acombination of 298A, 333A, and 334A), also referred to herein as thetriple Ala mutant. K334L increases binding to CD16. K322A results inreduced CDC activity; K326A or K326W enhances CDC activity. D265Aresults in reduced ADCC activity.

Stability variants are variants that show improved stability withrespect to, e.g., oxidation and deamidation.

A further type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. Such altering includes deletingone or more carbohydrate moieties found in the antibody, and/or addingone or more glycosylation sites that are not present in the antibody.Glycosylation variants that increase ADCC function are described, e.g.,in WO 2003/035835. See also US 2006/0067930.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US 2003/0157108 (Presta). See also US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisectingN-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fcregion of the antibody are referenced in, e.g., WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684 (Umana et al.).Antibodies with at least one galactose residue in the oligosaccharideattached to an Fc region of the antibody are reported, for example, inWO 1997/30087 (Patel et al.). See, also, WO 1998/58964 (Raju) and WO1999/22764 (Raju) concerning antibodies with altered carbohydrateattached to the Fc region thereof. See also US 2005/0123546 (Umana etal.); US 2004/0072290 (Umana et al.); US 2003/0175884 (Umana et al.); WO2005/044859 (Umana et al.); and US 2007/0111281 (Sondermann et al.) onantigen-binding molecules with modified glycosylation, includingantibodies with an Fc region containing N-linked oligosaccharides; andUS 2007/0010009 (Kanda et al.)

One preferred glycosylation antibody variant herein comprises an Fcregion wherein a carbohydrate structure attached to the Fc region hasreduced fucose or lacks fucose, which may improve ADCC function.Specifically, antibodies are contemplated herein that have reducedfusose relative to the amount of fucose on the same antibody produced ina wild-type CHO cell. That is, they are characterized by having a loweramount of fucose than they would otherwise have if produced by nativeCHO cells. Preferably the antibody is one wherein less than about 10% ofthe N-linked glycans thereon comprise fucose, more preferably whereinless than about 5% of the N-linked glycans thereon comprise fucose, andmost preferably, wherein none of the N-linked glycans thereon comprisefucose, i.e., wherein the antibody is completely without fucose, or hasno fucose.

Such “defucosylated” or “fucose-deficient” antibodies may be produced,for example, by culturing the antibodies in a cell line such as thatdisclosed in, for example, US 2003/0157108; WO 2000/61739; WO2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778;WO2005/053742; US 2006/0063254; US 2006/0064781; US 2006/0078990; US2006/0078991; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); andYamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Examples of celllines producing defucosylated antibodies include Lec13 CHO cellsdeficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.249:533-545 (1986); US 2003/0157108 A1 (Presta) and WO 2004/056312 A1(Adams et al., especially at Example 11) and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8-knockout CHO cells(Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004)). See also Kandaet al., Biotechnol. Bioeng., 94: 680-8 (2006). US 2007/0048300(Biogen-IDEC) discloses a method of producing aglycosylatedFc-containing polypeptides, such as antibodies, having desired effectorfunction, as well as aglycosylated antibodies produced according to themethod and methods of using such antibodies as therapeutics, all beingapplicable herein. Additionally, U.S. Pat. No. 7,262,039 relates to apolypeptide having an alpha-1,3-fucosyltransferase activity, including amethod for producing a fucose-containing sugar chain using thepolypeptide.

See also US 2006/024304 (Gerngross et al.); U.S. Pat. No. 7,029,872(Gerngross); US 2004/018590 (Gerngross et al.); US 2006/034828(Gerngross et al.); US 2006/034830 (Gerngross et al.); US 2006/029604(Gerngross et al.); WO 2006/014679 (Gerngross et al.); WO 2006/014683(Gerngross et al.); WO 2006/014685 (Gerngross et al.); WO 2006/014725(Gerngross et al.); and WO 2006/014726 (Gerngross et al.) on recombinantglycoproteins and glycosylation variants that are applicable herein.

In another embodiment, the invention provides an antibody compositioncomprising the antibodies described herein having an Fc region, whereinabout 20-100% of the antibodies in the composition comprise a maturecore carbohydrate structure in the Fc region that lacks a fucose.Preferably, such composition comprises antibodies having an Fc regionthat has been altered to change one or more of the antibody-dependentcell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity(CDC), or pharmacokinetic properties of the antibody compared to awild-type IgG Fc sequence by substituting an amino acid selected fromthe group consisting of A, D, E, L, Q, T, and Y at any one or anycombination of positions of the Fc region selected from the groupconsisting of: 238, 239, 246, 248, 249, 250, 252, 254, 255, 256, 258,265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290,292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 309, 312, 314,315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337,338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 428,430, 434, 435, 437, 438, and 439.

The composition is more preferably one wherein the antibody furthercomprises an Fc substitution that is 268D or 326D or 333A together with334A, or 298A together with 333A, or 298A together with 334A, or 239Dtogether with 332E, or 239D together with 298A and 332E, or 239Dtogether with 268D and 298A and 332E, or 239D together with 268D and298A and 326A and 332A, or 239D together with 268D and 298A and 326A and332E, or 239D together with 268D and 283L and 298A and 332E, or 239Dtogether with 268D and 283L and 298A and 326A and 332E, or 239D togetherwith 330L and 332E, wherein the letter after the number in each of thesedesignations represents the changed amino acid at that position.

The composition is additionally preferably one wherein the antibodybinds an FcγRIII. The composition further is preferably such that theantibody has ADCC activity in the presence of human effector cells orhas increased ADCC activity in the presence of human effector cellscompared to the otherwise same antibody comprising a human wild-typeIgG1Fc region.

The composition is also preferably one wherein the antibody binds theFcγRIII with better affinity, or mediates ADCC more effectively, than aglycoprotein with a mature core carbohydrate structure including fucoseattached to the Fc region of the glycoprotein. In addition, thecomposition is preferably one wherein the antibody has been produced bya CHO cell, preferably a Lec13 cell. The composition is also preferablyone wherein the antibody has been produced by a mammalian cell lacking afucosyltransferase gene, more preferably the FUT8 gene.

In one aspect, the composition is one wherein the antibody is free ofbisecting N-acetylglucosamine (GlcNAc) attached to the mature corecarbohydrate structure. Alternatively, the composition is such that theantibody has bisecting GlcNAc attached to the mature core carbohydratestructure.

In another aspect, the composition is one wherein the antibody has oneor more galactose residues attached to the mature core carbohydratestructure. Alternatively, the composition is such that the antibody isfree of one or more galactose residues attached to the mature corecarbohydrate structure.

In a further aspect, the composition is one wherein the antibody has oneor more sialic acid residues attached to the mature core carbohydratestructure. Alternatively, the composition is such that the antibody isfree of one or more sialic acid residues attached to the mature corecarbohydrate structure.

This composition preferably comprises at least about 2% afucosylatedantibodies. The composition more preferably comprises at least about 4%afucosylated antibodies. The composition still more preferably comprisesat least about 10% afucosylated antibodies. The composition even morepreferably comprises at least about 19% afucosylated antibodies. Thecomposition most preferably comprises about 100% afucosylatedantibodies.

Immunoconjugates

The invention also pertains to immunoconjugates, or antibody-drugconjugates (ADC), comprising an antibody conjugated to a cytotoxic agentsuch as a chemotherapeutic agent, a drug, a growth-inhibitory agent, atoxin (e.g., an enzymatically active toxin of bacterial, fungal, plant,or animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, e.g., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos, Anticancer Research19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drug Del. Rev.26:151-172 (1997); and U.S. Pat. No. 4,975,278) allows targeted deliveryof the drug moiety to tumors, and intracellular accumulation therein,where systemic administration of these unconjugated drug agents mayresult in unacceptable levels of toxicity to normal cells as well as thetumor cells sought to be eliminated (Baldwin et al., Lancet, 603-05(1986); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications, A. Pinchera et al. (eds), pp. 475-506 (1985)).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., Cancer Immunol. Immunother.,21:183-87 (1986)). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine. Toxins used in antibody-toxinconjugates include bacterial toxins such as diphtheria toxin, planttoxins such as ricin, small-molecule toxins such as geldanamycin(Mandler et al., J. Nat. Cancer Inst., 92(19):1573-1581 (2000); Mandleret al., Bioorganic & Med. Chem. Letters, 10:1025-1028 (2000); andMandler et al., Bioconjugate Chem., 13: 786-791 (2002)), maytansinoids(EP 1391213 and Liu et al., Proc. Natl. Acad. Sci. USA, 93: 8618-8623(1996)), and calicheamicin (Lode et al., Cancer Res., 58:2928 (1998);and Hinman et al., Cancer Res., 53:3336-3342 (1993)). Without beinglimited to any one theory, the toxins may exert their cytotoxic andcytostatic effects by mechanisms including tubulin binding, DNA binding,or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive orless active when conjugated to large antibodies or protein receptorligands.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, for example, WO1994/11026.

Conjugates of an antibody and one or more small-molecule toxins, such asa calicheamicin, maytansinoid, trichothecene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

In the ADCs of the invention, an antibody (Ab) is conjugated to one ormore drug moieties (D), e.g., about 1 to about 20 drug moieties perantibody, through a linker (L). The ADC of Formula I may be prepared byseveral routes, employing organic chemistry reactions, conditions, andreagents known to those skilled in the art, including: (1) reaction of anucleophilic group of an antibody with a bivalent linker reagent, toform Ab-L, via a covalent bond, followed by reaction with a drug moietyD; and (2) reaction of a nucleophilic group of a drug moiety with abivalent linker reagent, to form D-L, via a covalent bond, followed byreaction with the nucleophilic group of an antibody.Ab-(L-D)_(p)  Formula I

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side-chain amine groups, e.g., lysine,(iii) side-chain thiol groups, e.g., cysteine, and (iv) sugar hydroxylor amino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.,cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol.

Antibody-drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g., withperiodate oxidizing reagents, to form aldehyde or ketone groups that mayreact with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g., by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either galactose oxidase or sodium meta-periodate mayyield carbonyl (aldehyde and ketone) groups in the protein that canreact with appropriate groups on the drug (Domen et al., J. Chromatog.,510: 293-302 (1990)). In another embodiment, proteins containingN-terminal serine or threonine residues can react with sodiummeta-periodate, resulting in production of an aldehyde in place of thefirst amino acid (Geoghegan and Stroh, Bioconjugate Chem., 3:138-146(1992) and U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with adrug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; and (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide that does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

The ADCs herein are optionally prepared with cross-linker reagents, suchas, for example, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA,SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), which are commerciallyavailable (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog.

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water-soluble polymers. Non-limitingexamples of water-soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients, or stabilizers (Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low-molecular-weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose, or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™, orpolyethylene glycol (PEG).

A further formulation and delivery method herein involves thatdescribed, for example, in WO 2004/078140, including the ENHANZE™ drugdelivery technology (Halozyme Inc.). This technology is based on arecombinant human hyaluronidase (rHuPH20). rHuPH20 is a recombinant formof the naturally occurring human enzyme approved by the FDA thattemporarily clears space in the matrix of tissues such as skin. That is,the enzyme has the ability to break down hyaluronic acid (HA), thespace-filling “gel”-like substance that is a major component of tissuesthroughout the body. This clearing activity is expected to allow rHuPH20to improve drug delivery by enhancing the entry of therapeutic moleculesthrough the subcutaneous space. Hence, when combined or co-formulatedwith certain injectable drugs, this technology can act as a “molecularmachete” to facilitate the penetration and dispersion of these drugs bytemporarily opening flow channels under the skin. Molecules as large as200 nanometers may pass freely through the perforated extracellularmatrix, which recovers its normal density within approximately 24 hours,leading to a drug delivery platform that does not permanently alter thearchitecture of the skin.

Hence, the present invention includes a method of delivering an antibodyherein to a tissue containing excess amounts of glycosaminoglycan,comprising administering a hyaluronidase glycoprotein (sHASEGP) (thisprotein comprising a neutral active soluble hyaluronidase polypeptideand at least one N-linked sugar moiety, wherein the N-linked sugarmoiety is covalently attached to an asparagine residue of thepolypeptide) to the tissue in an amount sufficient to degradeglycosaminoglycans sufficiently to open channels less than about 500nanometers in diameter; and administering the antibody to the tissuecomprising the degraded glycosaminoglycans.

In another embodiment, the invention includes a method for increasingthe diffusion of an antibody herein that is administered to a subjectcomprising administering to the subject a sHASEGP polypeptide in anamount sufficient to open or to form channels smaller than the diameterof the antibody and administering the antibody, whereby the diffusion ofthe therapeutic substance is increased. The sHASEGP and antibody may beadministered separately or simultaneously in one formulation, andconsecutively in either order or at the same time.

Exemplary anti-LTα antibody formulations are described in WO 1998/56418,which include a liquid multidose formulation comprising the anti-LTαantibody at 40 mg/mL, 25 mM acetate, 150 mM trehalose, 0.9% benzylalcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf life oftwo years storage at 2-8° C. Another anti-LTα formulation of interestcomprises 10 mg/mL antibody in 9.0 mg/mL sodium chloride, 7.35 mg/mLsodium citrate dihydrate, 0.7 mg/mL POLYSORBATE 80™ surfactant, andSterile Water for Injection, pH 6.5. Yet another aqueous pharmaceuticalformulation comprises 10-30 mM sodium acetate from about pH 4.8 to aboutpH 5.5, preferably at pH 5.5, POLYSORBATE as a surfactant in an amountof about 0.01-0.1% v/v, trehalose at an amount of about 2-10% w/v, andbenzyl alcohol as a preservative (U.S. Pat. No. 6,171,586). Lyophilizedformulations adapted for subcutaneous administration are described, forexample, in WO 1997/04801 and U.S. Pat. No. 6,267,958 (Andya et al.).Such lyophilized formulations may be reconstituted with a suitablediluent to a high protein concentration and the reconstitutedformulation may be administered subcutaneously to the mammal to betreated herein.

Crystallized forms of the antibody are also contemplated. See, forexample, US 2002/0136719 (Shenoy et al.).

The formulation herein may also contain more than one active compound (asecond medicament as noted above) as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. For example, it may bedesirable to further provide a cytotoxic agent, chemotherapeutic agent,cytokine antagonist, integrin antagonist, or immunosuppressive agent(e.g., one that acts on T cells, such as cyclosporin or an antibody thatbinds T cells, e.g., one that binds LFA-1). The type and effectiveamounts of such second medicaments depend, for example, on the amount ofantibody present in the formulation, the type of disease or disorder ortreatment, the clinical parameters of the subjects, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as described herein or from about 1 to 99% of theheretofore employed dosages. The preferred such medicaments are notedabove.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug-delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are disclosed, forexample, in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in-vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Uses

An antibody of the present invention may be used in, for example, invitro, ex vivo, and in vivo therapeutic methods. Antibodies of theinvention can be used as an antagonist to partially or fully block thespecific LTα activity in vitro, ex vivo, and/or in vivo. Moreover, atleast some of the antibodies of the invention can neutralize antigenactivity from other species. Accordingly, the antibodies of theinvention can be used to inhibit a specific antigen activity, e.g., in acell culture containing the antigen, in human subjects, or in othermammalian subjects having the antigen with which an antibody of theinvention cross-reacts (e.g., chimpanzee, baboon, marmoset, cynomolgus,rhesus, pig, or mouse). In one embodiment, the antibody of the inventioncan be used for inhibiting antigen activities by contacting the antibodywith the antigen such that antigen activity is inhibited. Preferably,the antigen is a human protein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor inhibiting an antigen in a subject suffering from a disorder inwhich the antigen activity is detrimental, comprising administering tothe subject an antibody of the invention such that the antigen activityin the subject is inhibited. Preferably, the antigen is a human proteinmolecule and the subject is a human subject. Alternatively, the subjectcan be a mammal expressing the antigen to which an antibody of theinvention binds. Still further, the subject can be a mammal into whichthe antigen has been introduced (e.g., by administration of the antigenor by expression of an antigen transgene). An antibody of the inventioncan be administered to a human subject for therapeutic purposes.Moreover, an antibody of the invention can be administered to anon-human mammal expressing an antigen with which the immunoglobulincross-reacts (e.g., a primate, pig, or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). The antibodies of the invention can be used totreat, inhibit, delay progression of, prevent/delay recurrence of,ameliorate, or prevent autoimmune diseases, disorders, or conditions asdefined herein.

In one aspect, a blocking antibody of the invention is specific to aligand antigen, and inhibits the antigen activity by blocking orinterfering with the ligand-receptor interaction involving the ligandantigen, thereby inhibiting the corresponding signal pathway and othermolecular or cellular events. The invention also featuresreceptor-specific antibodies that do not necessarily prevent ligandbinding but interfere with receptor activation, thereby inhibiting anyresponses that would normally be initiated by the ligand binding. Theinvention also encompasses antibodies that either preferably orexclusively bind to ligand-receptor complexes. An antibody of theinvention can also act as an agonist of a particular antigen receptor,thereby potentiating, enhancing, or activating either all or partialactivities of the ligand-mediated receptor activation.

The antibody may be a naked antibody or alternatively is conjugated withanother molecule, such as a cytotoxic agent. The antibody is preferablyadministered intravenously or subcutaneously, most preferablysubcutaneously.

In one embodiment, the subject has never been previously treated withdrug(s), such as immunosuppressive agent(s), to treat the disorder, andin a particular embodiment has never been previously treated with a TNFantagonist. In an alternative embodiment, the subject has beenpreviously treated with drug(s) to treat the disorder, including with aTNF antagonist.

In a still further aspect, the patient has relapsed with the disorder.In an alternative embodiment, the patient has not relapsed with thedisorder.

In another aspect, the antibody herein is the only medicamentadministered to the subject to treat the disorder. In an alternativeaspect, the antibody herein is one of the medicaments used to treat thedisorder.

In a further aspect, the subject only has RA as an autoimmune disorder.Alternatively, the subject only has MS as an autoimmune disorder. Stillalternatively, the subject only has lupus, or ANCA-associatedvasculitis, or Sjögren's syndrome as an autoimmune disorder. In allcases autoimmune disorder is defined above.

In a still further embodiment, the subject has an abnormal level of oneor more regulatory cytokines, anti-nuclear antibodies (ANA),anti-rheumatoid factor (RF) antibodies, creatinine, blood urea nitrogen,anti-endothelial antibodies, anti-neutrophil cytoplasmic antibodies(ANCA), infiltrating CD20 cells, anti-double stranded DNA (dsDNA)antibodies, anti-Sm antibodies, anti-nuclear ribonucleoproteinantibodies, anti-phospholipid antibodies, anti-ribosomal P antibodies,anti-Ro/SS-A antibodies, anti-Ro antibodies, anti-La antibodies,antibodies directed against Sjögren's-associated antigen A or B (SS-A orSS-B), antibodies directed against centromere protein B (CENP B) orcentromere protein C (CENP C), autoantibodies to ICA69, anti-Smithantigen (Sm) antibodies, anti-nuclear ribonucleoprotein antibodies,anti-ribosomal P antibodies, autoantibodies staining the nuclear orperinuclear zone of neutrophils (pANCA), anti-Saccharomyces cerevisiaeantibodies, cross-reactive antibodies to GM1 ganglioside or GQ1bganglioside, anti-acetylcholine receptor (AchR), anti-AchR subtype, oranti-muscle specific tyrosine kinase (MuSK) antibodies, serumanti-endothelial cell antibodies, IgG or anti-desmoglein (Dsg)antibodies, anti-centromere, anti-topoisomerase-1 (Scl-70), anti-RNApolymerase or anti-U3-ribonucleoprotein (U3-RNP) antibodies,anti-glomerular basement membrane (GBM) antibodies, anti-glomerularbasement membrane (GBM) antibodies, anti-mitochondrial (AMA) oranti-mitochondrial M2 antibodies, anti-thyroid peroxidase (TPO),anti-thyroglobin (TG) or anti-thyroid stimulating hormone receptor(TSHR) antibodies, anti-nucleic (AN), anti-actin (AA) or anti-smoothmuscle antigen (ASM) antibodies, IgA anti-endomysial, IgA anti-tissuetransglutaminase, IgA anti-gliadin or IgG anti-gliadin antibodies,anti-CYP21A2, anti-CYP11A1 or anti-CYP17 antibodies,anti-ribonucleoprotein (RNP), or myosytis-specific antibodies,anti-myelin associated glycoprotein (MAG) antibodies, anti-hepatitis Cvirus (HCV) antibodies, anti-GM1 ganglioside, anti-sulfate-3-glycuronylparagloboside (SGPG), or IgM anti-glycoconjugate antibodies, IgManti-ganglioside antibody, anti-thyroid peroxidase (TPO),anti-thyroglobin (TG) or anti-thyroid stimulating hormone receptor(TSHR) antibodies, anti-myelin basic protein or anti-myelinoligodendrocytic glycoprotein antibodies, IgM rheumatoid factorantibodies directed against the Fc portion of IgG, anti-Factor VIIIantibodies, or a combination thereof.

The parameters for assessing efficacy or success of treatment of anautoimmune disorder (which includes an autoimmune-related disease) willbe known to the physician of skill in the appropriate disease.Generally, the physician of skill will look for reduction in the signsand symptoms of the specific disease. The following are by way ofexamples.

In one embodiment, the methods and compositions of the invention areuseful to treat RA. RA is characterized by inflammation of multiplejoints, cartilage loss, and bone erosion that leads to joint destructionand ultimately reduced joint function. Additionally, since RA is asystemic disease, it can have effects in other tissues such as thelungs, eyes, and bone marrow.

If the subject has RA, which is a preferred indication herein,preferably the antibodies herein induce a major clinical response in thesubject.

The antibodies herein can be used as first-line therapy in patients withearly RA (i.e., methotrexate (MTX) naive), or in combination with, e.g.,MTX or cyclophosphamide. Alternatively, the antibodies can be used intreatment as second-line therapy for patients who were, for example,refractory to DMARD and/or TNF inhibitor, and/or MTX, in combinationwith, e.g., MTX. The LTα-binding antibodies can also be administered incombination with B-cell mobilizing agents such as integrin antibodiesthat mobilize B cells into the bloodstream for more effective killing.The LTβ-binding antibodies are useful to prevent and control jointdamage, delay structural damage, decrease pain associated withinflammation in RA, and generally reduce the signs and symptoms inmoderate-to-severe RA. The RA patient can be treated with the LTαantibody prior to, after, or together with treatment with other drugsused in treating RA (see combination therapy described herein). Patientswho had previously failed DMARDs and/or had an inadequate response toMTX alone are, in one embodiment, treated with a LTα-binding antibody.In another embodiment, such patients are administered humanizedLTα-binding antibody plus cyclophosphamide or LTα-binding antibody plusMTX. Most preferably, the antibodies herein are administered with MTXfor treatment of RA, and not with high-dose steroids.

One method of evaluating treatment efficacy in RA is based on AmericanCollege of Rheumatology (ACR) criteria, which are used to measure thepercentage of improvement in tender and swollen joints, among otherthings. The RA patient can be scored at, for example, ACR 20 (20 percentimprovement) compared with no antibody treatment (e.g, baseline beforetreatment) or treatment with placebo. Other ways of evaluating theefficacy of antibody treatment include X-ray scoring such as the SharpX-ray score used to score structural damage such as bone erosion andjoint space narrowing. Patients can also be evaluated for the preventionof or improvement in disability based on Health Assessment Questionnaire(HAQ) score, AIMS score, or SF-36 at time periods during or aftertreatment. The ACR 20 criteria may include 20% improvement in bothtender (painful) joint count and swollen joint count plus a 20%improvement in at least three of five additional measures:

-   -   1. patient's pain assessment by visual analog scale (VAS),    -   2. patient's global assessment of disease activity (VAS),    -   3. physician's global assessment of disease activity (VAS),    -   4. patient's self-assessed disability measured by the Health        Assessment Questionnaire, and    -   5. acute phase reactants, CRP or ESR.

The ACR 50 and 70 are defined analogously. Preferably, the patient isadministered an amount of an LTα-binding antibody of the inventioneffective to achieve at least a score of ACR 20, preferably at least ACR30, more preferably at least ACR 50, even more preferably at least ACR70, and most preferably at least ACR 75.

Psoriatic arthritis has unique and distinct radiographic features. Forpsoriatic arthritis, joint erosion and joint space narrowing can beevaluated by the Sharp score as well. The antibodies disclosed hereincan be used to prevent the joint damage as well as reduce disease signsand symptoms of the disorder.

Yet another aspect of the invention is a method of treating lupus,including systemic lupus erythematosus (SLE) and lupus nephritis, byadministering to the subject having such disease an effective amount ofan antibody of the invention. For example, SLEDAI scores provide anumerical quantitation of disease activity. The SLEDAI is a weightedindex of 24 clinical and laboratory parameters known to correlate withdisease activity, with a numerical range of 0-103. See Gescuk and Davis,Current Opinion in Rheumatology, 14: 515-521 (2002). Antibodies todouble-stranded DNA are believed to cause renal flares and othermanifestations of lupus. Patients undergoing antibody treatment can bemonitored for time to renal flare, which is defined as a significant,reproducible increase in serum creatinine, urine protein, or blood inthe urine. Alternatively or in addition, patients can be monitored forlevels of antinuclear antibodies and antibodies to double-stranded DNA.Treatments for SLE include high-dose corticosteroids and/orcyclophosphamide (HDCC).

Spondyloarthropathies are a group of disorders of the joints, includingankylosing spondylitis, psoriatic arthritis, and Crohn's disease.Treatment success can be determined by validated patient and physicianglobal assessment measuring tools.

Various medications are used to treat psoriasis; treatment differsdirectly in relation to disease severity. Patients with a more mild formof psoriasis typically utilize topical treatments, such as topicalsteroids, anthralin, calcipotriene, clobetasol, and tazarotene, tomanage the disease, while patients with moderate and severe psoriasisare more likely to employ systemic (methotrexate, retinoids,cyclosporine, PUVA, and UVB) therapies. Tars are also used. Thesetherapies are disadvantageous due to safety concerns, time-consumingregimens, and/or inconvenient processes of treatment. Furthermore, somerequire expensive equipment and dedicated space in the office setting.Such systemic medications can produce serious side effects, includinghypertension, hyperlipidemia, bone-marrow suppression, liver disease,kidney disease, and gastrointestinal upset. Also, the use ofphototherapy can increase the incidence of skin cancers. In addition tothe inconvenience and discomfort associated with the use of topicaltherapies, phototherapy and such systemic treatments also requirecycling patients on and off therapy and monitoring lifetime exposure dueto their side effects.

Treatment efficacy for psoriasis is assessed by monitoring changes inclinical signs and symptoms of the disease, including Physician's GlobalAssessment (PGA) changes, Psoriasis Area and Severity Index (PASI)scores, and Psoriasis Symptom Assessment (PSA), compared with thebaseline condition. The patient can be measured periodically throughouttreatment on the Visual Analog Scale (VAS) used to indicate the degreeof itching experienced at specific time points.

Assays

Ligand/receptor binding studies may be carried out in any known assaymethod, such as competitive-binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Cell-based assays and animalmodels can be used to understand the interaction between the ligands andreceptors identified herein and the development and pathogenesis of theconditions and diseases referred to herein.

In one approach, mammalian cells may be transfected with the ligands orreceptors described herein, and the ability of the antibody herein toinhibit binding or activity is analyzed. Suitable cells can betransfected with the desired gene, and monitored for activity. Suchtransfected cell lines can then be used to test the ability of antibody,for example, to modulate LTαβ complex expression of the cells.

In addition, primary cultures derived from transgenic animals can beused in the cell-based assays. Techniques to derive continuous celllines from transgenic animals are well known in the art. See, e.g.,Small et al., Mol. Cell. Biol., 5:642-648 (1985).

One suitable cell-based assay is the addition of epitope-tagged ligand(e.g., LTα) to cells that have or express the respective receptor, andthe analysis of binding (in the presence or absence of prospectiveantibodies) by FACS staining with anti-tag antibody. In another assay,the ability of the antibody herein to inhibit the expression of LTαβcomplex on cells expressing such complex is assayed. For example,suitable expressing cell lines are cultured in the presence or absenceof prospective antibodies and the modulation of LTαβ complex expressioncan be measured by ³H-thymidine incorporation or cell number.

The results of the cell-based in vitro assays can be further verifiedusing in vivo animal models. Many animal models can be used to test theefficacy of the antibodies identified herein in relation to, forinstance, immune-related disease. The in vivo nature of such modelsmakes them particularly predictive of responses in human patients.Animal models of immune-related diseases include both non-recombinantand recombinant (transgenic) animals. Non-recombinant animal modelsinclude, for example, rodent, e.g., murine models. Such models can begenerated by introducing cells into syngeneic mice using standardtechniques, e.g. subcutaneous injection, tail-vein injection, spleenimplantation, intraperitoneal implantation, and implantation under therenal capsule.

Graft-versus-host disease is an example of a disease for which an animalmodel has been designed. Graft-versus-host disease occurs whenimmunocompetent cells are transplanted into immunosuppressed or tolerantpatients. The donor cells recognize and respond to host antigens. Theresponse can vary from life-threatening severe inflammation to mildcases of diarrhea and weight loss. Graft-versus-host disease modelsprovide a means of assessing T-cell reactivity against MHC antigens andminor transplant antigens. A suitable procedure is described in detailin Current Protocols in Immunology, Eds. Cologan et al., (John Wiley &Sons, Inc., 1994), unit 4.3.

An animal model for skin allograft rejection tests the ability of Tcells to mediate in vivo tissue destruction to measure their role inanti-viral immunity, and is described, for example, in Current Protocolsin Immunology, supra, unit 4.4. Other transplant rejection models usefulto test the antibodies herein include the allogeneic heart-transplantmodels described by Tanabe et al., Transplantation, 58:23 (1994) andTinubu et al., J. Immunol., 4330-4338 (1994).

Animal models for delayed-type hypersensitivity provide an assay ofcell-mediated immune function. Delayed-type hypersensitivity reactionsare a T-cell-mediated in vivo immune response characterized byinflammation that does not reach a peak until after a period of time haselapsed after challenge with an antigen. These reactions also occur intissue-specific autoimmune diseases such as MS and experimentalautoimmune encephalomyelitis (EAE, a model for MS). A suitable,exemplary model is described in detail in Current Protocols inImmunology, supra, unit 4.5.

The collagen-induced arthritis (CIA) model is considered a suitablemodel for studying potential drugs or biologics active in humanarthritis because of the many immunological and pathologicalsimilarities to human RA, the involvement of localized majorhistocompatibility, complete class-II-restricted T-helper lymphocyteactivation, and the similarity of histological lesions. Features of thisCIA model that are similar to that found in RA patients include: erosionof cartilage and bone at joint margins (as can be seen in radiographs),proliferative synovitis, and symmetrical involvement of small andmedium-sized peripheral joints in the appendicular, but not the axial,skeleton. Jamieson et al., Invest. Radiol. 20: 324-9 (1985).Furthermore, IL-1 and TNF-α appear to be involved in CIA as well as inRA. Joosten et al., J. Immunol. 163: 5049-5055 (1999). TNF-neutralizingantibodies, and separately, TNFR.Fc, reduced the symptoms of RA in thismodel (Williams et al., Proc. Natl. Acad. Sci. USA, 89:9784-9788 (1992);Wooley et al., J. Immunol., 151: 6602-6607 (1993)).

In this model for RA, type II collagen is purified from bovine articularcartilage (Miller, Biochemistry 11:4903 (1972)) and used to immunizedmice (Williams et al, Proc. Natl. Acad. Sci. USA, 91:2762 (1994)).Symptoms of arthritis include erythema and/or swelling of the limbs aswell as erosions or defects in cartilage and bone as determined byhistology. This widely used model is also described, for example, byHolmdahl et al., APMIS, 97:575 (1989), in Current Protocols inImmunology, supra, units 15.5, and in Issekutz et al., Immunology,88:569 (1996).

A model of asthma has been described in which antigen-induced airwayhyper-reactivity, pulmonary eosinophilia, and inflammation are inducedby sensitizing an animal with ovalbumin and challenging the animal withthe same protein delivered by aerosol. Animal models such as rodent andnon-human primate models exhibit symptoms similar to atopic asthma inhumans upon challenge with aerosol antigens. Suitable procedures to testthe antibodies herein for suitability in treating asthma include thosedescribed by Wolyniec et al., Am. J. Respir. Cell Mol. Biol., 18:777(1998).

Additionally, the antibodies herein can be tested in the SCID/SCID mousemodel for immune disorders. For example, as described by Schon et al.,Nat. Med., 3:183 (1997), the mice demonstrate histopathologic skinlesions resembling psoriasis. Another suitable model is the humanskin/SCID mouse chimera prepared as described by Nickoloff et al., Am.J. Path., 146:580 (1995).

Dosage

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with asecond medicament as noted below) will depend, for example, on the typeof disease to be treated, the type of antibody, the severity and courseof the disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The dosage is preferably efficacious for the treatment ofthat indication while minimizing toxicity and side effects.

The antibody is suitably administered to the patient at one time or overa series of treatments. Depending on the type and severity of thedisease, about 1 μg/kg to 500 mg/kg (preferably about 0.1 mg/kg to 400mg/kg) of antibody is an initial candidate dosage for administration tothe patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 500 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment is sustained untila desired suppression of disease symptoms occurs. One exemplary dosageof the antibody would be in the range from about 0.05 mg/kg to about 400mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg or 50 mg/kg or 100 mg/kg or 300 mg/kg or 400 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e.g., every week or every three weeks(e.g., such that the patient receives from about two to about twenty,e.g., about six doses of the antibody). An initial higher loading dose,followed by one or more lower doses, may be administered. An exemplarydosing regimen comprises administering an initial loading dose of about4 to 500 mg/kg, followed by a weekly maintenance dose of about 2 to 400mg/kg of the antibody. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

For the treatment of an autoimmune disorder, the therapeuticallyeffective dosage will typically be in the range of about 50 mg/m² toabout 3000 mg/m², preferably about 50 to 1500 mg/m², more preferablyabout 50-1000 mg/m². In one embodiment, the dosage range is about125-700 mg/m². For treating RA, in one embodiment, the dosage range forthe humanized antibody is about 50 mg/m² or 125 mg/m² (equivalent toabout 200 mg/dose) to about 1000 mg/m², given in two doses, e.g., thefirst dose of about 200 mg is administered on day one followed by asecond dose of about 200 mg on day 15. In different embodiments, thedosage is about any one of 50 mg/dose, 80 mg/dose, 100 mg/dose, 125mg/dose, 150 mg/dose, 200 mg/dose, 250 mg/dose, 275 mg/dose, 300mg/dose, 325 mg/dose, 350 mg/dose, 375 mg/dose, 400 mg/dose, 425mg/dose, 450 mg/dose, 475 mg/dose, 500 mg/dose, 525 mg/dose, 550mg/dose, 575 mg/dose, or 600 mg/dose, or 700 mg/dose, or 800 mg/dose, or900 mg/dose, or 1000 mg/dose, or 1500 mg/dose.

In treating disease, the LTα-binding antibodies of the invention can beadministered to the patient chronically or intermittently, as determinedby the physician of skill in the disease.

A patient administered a drug by intravenous infusion or subcutaneouslymay experience adverse events such as fever, chills, burning sensation,asthenia, and headache. To alleviate or minimize such adverse events,the patient may receive an initial conditioning dose(s) of the antibodyfollowed by a therapeutic dose. The conditioning dose(s) will be lowerthan the therapeutic dose to condition the patient to tolerate higherdosages.

The antibodies herein may be administered at a frequency that is withinthe skill and judgment of the practicing physician, depending on variousfactors noted above, for example, the dosing amount. This frequencyincludes twice a week, three times a week, once a week, bi-weekly, oronce a month, In a preferred aspect of this method, the antibody isadministered no more than about once every other week, more preferablyabout once a month.

Route of Administration

The antibodies used in the methods of the invention (as well as anysecond medicaments) are administered to a subject or patient, includinga human patient, in accord with suitable methods, such as those known tomedical practitioners, depending on many factors, including whether thedosing is acute or chronic. These routes include, for example,parenteral, intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by subcutaneous,intramuscular, intra-arterial, intraperitoneal, intrapulmonary,intracerebrospinal, intra-articular, intrasynovial, intrathecal,intralesional, or inhalation routes (e.g., intranasal). Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theantibody is suitably administered by pulse infusion, particularly withdeclining doses of the antibody. Preferred routes herein are intravenousor subcutaneous administration, most preferably subcutaneous.

In one embodiment, the antibody herein is administered by intravenousinfusion, and more preferably with about 0.9 to 20% sodium chloridesolution as an infusion vehicle.

Combination Therapy

In any of the methods herein, one may administer to the subject orpatient along with the antibody herein an effective amount of a secondmedicament (where the antibody herein is a first medicament), which isanother active agent that can treat the condition in the subject thatrequires treatment; for example, if the condition is an autoimmunedisorder, the second medicament is a drug that can treat the autoimmunedisorder, alone or with another active agent. For treatment ofautoimmune disorders, the second medicament includes, for example, achemotherapeutic agent, an immunosuppressive agent, an antagonist (suchas an antibody) that binds a B-cell surface marker such as rituximab orhumanized 2H7, a BAFF antagonist, a DMARD, a cytotoxic agent, anintegrin antagonist such as a CD11 or CD18 antagonist including CD11a orCD18 antibodies, e.g., efalizumab (RAPTIVA®), a NSAID, a cytokineantagonist, such as a TNF antagonist, an anti-rheumatic agent, a musclerelaxant, a narcotic, an analgesic, an anesthetic, a sedative, a localanesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic,a corticosteriod, an anabolic steroid, an erythropoietin, an immunizingagent, an immunoglobulin, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine, a cytokine, cells for repressing B-cellautoantibody secretion as set forth in WO 2005/027841, a hyaluronidaseglycoprotein as an active delivery vehicle as set forth in, for example,WO 2004/078140, or a combination thereof.

For treatment of proliferative diseases such as cancer, the secondmedicament would include, for example, chemotherapeutic agents,hormones, cytotoxic agents, and other biologics such as antibodies toHER-2, to VEGF, to B-cell surface antigens such as TAHO antigen (e.g.,US 2006/0251662), CD20, and CD22, and to EGF/EGF-R.

Preferably, such second medicament for autoimmune diseases is animmunosuppressive agent, an antagonist that binds a B-cell surfacemarker (such as an antibody, e.g., CD20 antibody or CD22 antibody), aBAFF antagonist, a DMARD, an integrin antagonist, a hyaluronidaseglycoprotein (as an active delivery vehicle), a NSAID, a cytokineantagonist, more preferably a TNF (e.g., TNF-alpha), CD11, or CD18antagonist, or a combination thereof. More preferably, it ismethotrexate, a TNF antagonist, an antagonist to a B-cell surfacemarker, or a DMARD.

The type of such second medicament depends on various factors, includingthe type of autoimmune disorder, the severity of the disease, thecondition and age of the patient, the type and dose of first medicamentemployed, etc.

The antibodies herein can be administered concurrently, sequentially, oralternating with the second medicament or upon non-responsiveness withother therapy. Thus, the combined administration of a second medicamentincludes co-administration (concurrent administration), using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) medicaments simultaneously exert theirbiological activities. All these second medicaments may be used incombination with each other or by themselves with the first medicament,so that the expression “second medicament” as used herein does not meanit is the only medicament besides the first medicament, respectively.Thus, the second medicament need not be one medicament, but mayconstitute or comprise more than one such drug.

These second medicaments as set forth herein are generally used in thesame dosages and with the same administration routes as the firstmedicaments, or from about 1 to 99% of the dosages of the firstmedicaments. If such second medicaments are used at all, preferably,they are used in lower amounts than if the first medicament were notpresent, especially in subsequent dosings beyond the initial dosing withthe first medicament, so as to eliminate or reduce side effects causedthereby.

For the treatment of RA, for example, the patient can be treated with anantibody of the invention in conjunction with any one or more of thefollowing drugs: integrin antagonists, DMARDs (e.g., MTX), NSAIDs,cytokine inhibitors such as HUMIRA™ (adalimumab; Abbott Laboratories),ARAVA® (leflunomide), REMICADE® (infliximab; Centocor Inc., of Malvern,Pa.), ENBREL® (etanercept; Immunex, WA), corticosteroids, or COX-2inhibitors. DMARDs commonly used in RA include hydroxycloroquine,sulfasalazine, methotrexate, leflunomide, etanercept, infliximab,azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular),minocycline, cyclosporine, or Staphylococcal protein A immunoadsorption.Adalimumab is a human monoclonal antibody that binds to TNFα. Infliximabis a chimeric monoclonal antibody that binds to TNFα. Etanercept is an“immunoadhesin” fusion protein consisting of the extracellular ligandbinding portion of the human 75 kD (p75) tumor necrosis factor receptor(TNFR) linked to the Fc portion of a human IgG1. For conventionaltreatment of RA, see, e.g., American College of RheumatologySubcommittee on Rheumatoid Arthritis Guidelines, Arthritis & Rheumatism46(2): 328-346 (2002). In a specific embodiment, the RA patient istreated with an LTα antibody of the invention in conjunction with MTX.An exemplary dosage of MTX is about 7.5-25 mg/kg/wk. MTX can beadministered orally and subcutaneously.

For the treatment of ankylosing spondylitis, psoriatic arthritis, andCrohn's disease, the patient can be treated with an antibody of theinvention in conjunction with, for example, REMICADE® (infliximab; fromCentocor Inc., Malvern, Pa.) or ENBREL® (etanercept; Amgen, Calif.).

Treatments for SLE include high-dose corticosteroids and/orcyclophosphamide (HDCC) in conjunction with the antibodies herein.

For the treatment of psoriasis, patients can be administered aLTβ-binding antibody in conjunction with topical treatments, such astopical steroids, anthralin, calcipotriene, clobetasol, and tazarotene,or with MTX, retinoids, cyclosporine, PUVA and UVB therapies, andintegrin antagonists such as anti-CD11a or anti-CD 18 antibodies,including, e.g., efalizumab (RAPTIVA®). In one embodiment, the psoriasispatient is treated with the LTα-binding antibody sequentially orconcurrently with cyclosporine or with efalizumab.

In certain embodiments, an immunoconjugate comprising the antibodyherein conjugated with a cytotoxic agent is administered to the patient.In some embodiments, the immunoconjugate and/or antigen to which it isbound is/are internalized by the cell, resulting in increasedtherapeutic efficacy of the immunoconjugate in killing the target cellto which it binds. In one embodiment, the cytotoxic agent targets orinterferes with nucleic acid in the target cell. Examples of suchcytotoxic agents include any of the chemotherapeutic agents noted herein(such as a maytansinoid or a calicheamicin), a radioactive isotope, aribonuclease, or a DNA endonuclease.

Articles of Manufacture

In another embodiment of the invention, articles of manufacturecontaining materials useful for the treatment of the disorders describedabove are provided. In one aspect, the article of manufacture comprises(a) a container comprising the antibodies herein (preferably thecontainer comprises the antibody and a pharmaceutically acceptablecarrier or diluent within the container); and (b) a package insert withinstructions for treating the disorder in a patient.

In a preferred embodiment, the article of manufacture herein furthercomprises a container comprising a second medicament, wherein theantibody is a first medicament. This article further comprisesinstructions on the package insert for treating the patient with thesecond medicament, in an effective amount. The second medicament may beany of those set forth above, with an exemplary second medicament beingan antagonist binding to a B-cell surface marker (e.g., a CD20antibody), a BAFF antagonist, an immunosuppressive agent, including MTXand corticosteroids, an integrin antagonist, a cytokine antagonist suchas a TNF, CD11, or CD18 antagonist, or a combination thereof. Thepreferred second medicaments are a steroid, a cytokine antagonist,and/or an immunosuppressive agent.

In this aspect, the package insert is on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds or contains a compositionthat is effective for treating the disorder in question and may have asterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is theantibody herein. The label or package insert indicates that thecomposition is used for treating the particular disorder in a patient orsubject eligible for treatment with specific guidance regardingadministration of the compositions to the patients, including dosingamounts and intervals of antibody and any other medicament beingprovided. Package insert refers to instructions customarily included incommercial packages of therapeutic products that contain informationabout the indications, usage, dosage, administration,contra-indications, and/or warnings concerning the use of suchtherapeutic products. The article of manufacture may further comprise anadditional container comprising a pharmaceutically acceptable diluentbuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution, and/or dextrose solution.The article of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

In a specific embodiment of the invention, an article of manufacture isprovided comprising, packaged together, a pharmaceutical compositioncomprising an antibody herein and a pharmaceutically acceptable carrierand a label stating that the antibody or pharmaceutical composition isindicated for treating patients with an autoimmune disease such as RA,MS, lupus, or IBD.

In a preferred embodiment the article of manufacture herein furthercomprises a container comprising a second medicament, wherein theantibody is a first medicament, and which article further comprisesinstructions on the package insert for treating the patient with thesecond medicament, in an effective amount. The second medicament may beany of those set forth above, with an exemplary second medicament beingthose set forth above, including, especially for RA treatment, animmunosuppressive agent, a corticosteroid, a DMARD, an integrinantagonist, a NSAID, a cytokine antagonist, a bisphosphonate, or acombination thereof, more preferably a DMARD, NSAID, cytokineantagonist, integrin antagonist, or immunosuppressive agent. Mostpreferably, the second medicament is methotrexate or a DMARD if thedisease is RA.

In another aspect, the invention provides a method for packaging ormanufacturing an antibody herein or a pharmaceutical composition thereofcomprising combining in a package the antibody or pharmaceuticalcomposition and a label stating that the antibody or pharmaceuticalcomposition is indicated for treating patients with an autoimmunedisease such as RA, MS, lupus, or IBD.

Methods of Advertising

The invention herein also encompasses a method for advertising anantibody herein or a pharmaceutically acceptable composition thereofcomprising promoting, to a target audience, the use of the antibody orpharmaceutical composition thereof for treating a patient or patientpopulation with an autoimmune disease such as RA, MS, lupus, or IBD.

Advertising is generally paid communication through a non-personalmedium in which the sponsor is identified and the message is controlled.Advertising for purposes herein includes publicity, public relations,product placement, sponsorship, underwriting, and sales promotion. Thisterm also includes sponsored informational public notices appearing inany of the print communications media designed to appeal to a massaudience to persuade, inform, promote, motivate, or otherwise modifybehavior toward a favorable pattern of purchasing, supporting, orapproving the invention herein.

The advertising and promotion of the treatment methods herein may beaccomplished by any means. Examples of advertising media used to deliverthese messages include television, radio, movies, magazines, newspapers,the internet, and billboards, including commercials, which are messagesappearing in the broadcast media. Advertisements also include those onthe seats of grocery carts, on the walls of an airport walkway, and onthe sides of buses, or heard in telephone hold messages or in-store PAsystems, or anywhere a visual or audible communication can be placed,generally in public places. More specific examples of promotion oradvertising means include television, radio, movies, the internet suchas webcasts and webinars, interactive computer networks intended toreach simultaneous users, fixed or electronic billboards and otherpublic signs, posters, traditional or electronic literature such asmagazines and newspapers, other media outlets, presentations orindividual contacts by, e.g., e-mail, phone, instant message, postal,courier, mass, or carrier mail, in-person visits, etc.

The type of advertising used will depend on many factors, for example,on the nature of the target audience to be reached, e.g., hospitals,insurance companies, clinics, doctors, nurses, and patients, as well ascost considerations and the relevant jurisdictional laws and regulationsgoverning advertising of medicaments. The advertising may beindividualized or customized based on user characterizations defined byservice interaction and/or other data such as user demographics andgeographical location.

The following are non-limiting examples of the methods and compositionsof the invention. It is understood that various other embodiments may bepracticed, given the general description provided above. The disclosuresof all citations in the specification are expressly incorporated hereinby reference.

EXAMPLE 1 Preparation of Anti-Human and Anti-Murine LTα MonoclonalAntibodies and Hamster-Murine Chimeras

Anti-human LTα monoclonal antibodies 2C8 and 3F12 were generated asfollows: BALB/c mice (Charles River Laboratories, Wilmington, Del.) werehyperimmunized by injection with 5 μg/dose of purified recombinant humanLTα expressed in E. coli (Genentech, Inc., South San Francisco, Calif.;Genentech Lot#8360-2B; see also Spriggs, “Tumor Necrosis Factor: BasicPrinciples and Preclinical Studies,” Biologic Therapy of Cancer, DeVitaet al., eds., J.B. Lippincott Company (1991) Ch. 16, pp. 354-377;Ruddle, Current Opinion in Immunology, 4:327-332 (1992); Wong et al.,“Tumor Necrosis Factor,” Human Monocytes, Academic Press (1989), pp.195-215; Aggarwal et al., Cytokines and Lipocortins in Inflammation andDifferentiation, Wiley-Liss, Inc. 1990, pp. 375-384; and Paul et al.,Ann. Rev. Immunol., 6:407-438 (1988)) in DETOX™ adjuvant (RIBIImmunoChem Research, Inc., Hamilton, Mo.). Specifically, 10 μg LTα3 wasmixed with 50 μL of the adjuvant and injected subcutaneously into therear footpads of mice (strain BALB/c) on days 1, 4, 15, 29, 41, 56, and71. Serum was collected on Days 15 and 56 for detection ofanti-LTα3-specific antibodies by binding ELISA.

On day 75, the animals were sacrificed, spleens were harvested, and3×10E7 cells were fused with 5×10E7 cells of the mouse myeloma lineX63-Ag8.653 using 50% polyethylene glycol (PEG) 4000 by an establishedprocedure (Oi and Herzenberg, in Selected Methods in CellularImmunology, B. Mishel and S. Schiigi, eds., (W.J.Freeman Co., SanFrancisco, Calif., 1980)). The fused cells were plated into 96-wellmicrotiter plates at a density of 2×10E5 cells/well containing HATmedium for selection (Littlefield, Science, 145:709 (1964)). After 12days, the supernatants were harvested and screened for antibodyproduction by direct ELISA. The supernatants harvested from eachhybridoma lineage were purified by affinity chromatography (Pharmaciafast protein liquid chromatography (FPLC); Pharmacia, Uppsala, Sweden).The purified antibody preparations were then sterile filtered (0.2-μmpore size; Nalgene, Rochester N.Y.) and stored at 4° C. inphosphate-buffered saline (PBS).

Anti-murine LTα monoclonal antibody S5H3 was generated as follows:Armenian hamsters were immunized with recombinant mouse LTα2β1 (R&DSystems Inc. (1008-LY)). The hamsters were immunized with 2 μg ofantigen by footpad for 12 injections, with an IP boost of 2 μg ofantigen once. The lymph nodes and spleen were fused by PEG treatment.Primary screening was done by ELISA on Immunogen-vs.Genentech-manufactured murine LTα-6His (murine LTα with six histidinesattached at the C-terminal end). Detection was done withperoxidase-conjugated AFFINIPURE™ goat anti-Armenian hamster IgG (H+L)(Jackson ImmunoResearch 127-035-160). Secondary screening was performedby FACS on irrelevant transfected HEK 293 cells vs. HEK 293 cells stablytransfected with murine LTαβ (under G418 selection). Detection wasperformed using R-phycoerythrin-conjugated AFFINIPURE™ F(ab′)2 goatanti-Armenian hamster IgG (H+L) (Jackson ImmunoResearch 127-116-160).The hybridoma from which this antibody was derived was deposited withATCC as Deposit No. PTA-7538 (hybridoma murine Lymphotoxin alpha2 beta1s5H3.2.2).

All anti-murine and anti-human LTα antibodies generated in theseexperiments were selected for recognition of LTα3. These were thenscreened for (a) blocking of binding of LTα3 to TNFR1 and TNFR11 byELISA, (b) binding of LTαβ by ELISA and FACS of cells stably transfectedwith LTα and LTβ, and (c) blocking of LTαβ interaction with the LTβreceptor by ELISA. Antibodies 3F12, 2C8, and S5H3 were selected becausethey all bound and neutralized LTα3 and LTαβ.

For use in the murine in vivo studies described below, the hamsteranti-murine LTα hybridoma was cloned as a hamster/mouse chimera. TotalRNA was extracted from S5H3 hybridoma cells using a RNEASY MINI™ kit(Qiagen) and the manufacturer's suggested protocol. RT-PCR wasaccomplished using a Qiagen ONE-STEP™ kit, and primers were designed toamplify the light- and heavy-chain variable domains and add restrictionenzyme sites for cloning.

For construction of the light chain of this chimeric antibody S5H3, twosequential cloning steps were used. In the first step, RT-PCR, ClaI andAscI sites were added, using primer set one, allowing cloning into apRK-based vector simply to amplify DNA for sequencing. After the DNAsequence of the light-chain variable domain was obtained, a round of PCRwas performed, using this plasmid as template, in order to add EcoRV andKpnI sites that are compatible with the expression vector.

For construction of the heavy chain of S5H3, likewise two cloning stepswere used. Specifically, RT-PCR was used to amplify cDNA and add ClaIand AscI sites for cloning into a vector for sequencing. Subsequently,this plasmid was used as template for PCR to add BsiWI and ApaI sitesthat are compatible with the expression vector.

For RT-PCR, the primers used were as follows:

S5H3 light-chain set one: 5′ primer: (SEQ ID NO: 56)GGA TCA TCG ATA CAR CTN GTV YTN CAN CAR TCN CC 3′ primer:(SEQ ID NO: 57) GGT AAC GGC GCG CCG YTC AGA AGA TGG TGG RAAS5H3 light-chain set two: 5′ primer: (SEQ ID NO: 58)GGA TCC GAT ATC CAG CTG GTA TTG ACC CAA TCT 3′ primer: (SEQ ID NO: 59)GGA TCA GGT ACC GCT GCC AAA AAC ACA CGA CCC S5H3 heavy chain set one: 5′primer: (SEQ ID NO: 60) TGA TAA TCG ATG ARG TNC CAT TTR GTN GAR 3′primer: (SEQ ID NO: 61) TAG TAA GGC GCG CCT GGT CAG GGA NCC NGA RTT CCAS5H3 heavy chain set two: 5′ primer: (SEQ ID NO: 62)TGA TCG CGT ACG CTG AGG TTC AAT TGG TTG AG 3′ primer: (SEQ ID NO: 63)TGA TCG TGG GCC CTT TGT TGT GGC TGA GGA GAC GGwherein R=A or G, Y=C or T, and N=all four nucleic acids A, G, C, and T.Purified PCR products for the light-chain variable domain were clonedinto a pRK mammalian cell expression vector (pRK.LPG2.mukappa)containing the murine kappa constant domain. For the heavy chain, PCRproducts were cloned into a pRK mammalian cell expression vector(pRK.LPG10.mulgG2a) encoding the murine CH1, hinge, CH2, and CH3 domainsof murine isotype IgG2a. These vectors are derivatives of previouslydescribed vectors for expression of IgGs in 293 cells (Shields et al.,J. Biol. Chem., 276:6591-6604 (2000) and Gorman et al., DNA Prot EngTech, 2:3-10 (1990)). For S5H3 chimeric antibody expression, theplasmids for the light and heavy chains were transiently co-transfectedinto 293 cells (an adenovirus-transformed human embryonic kidney cellline (Graham et al., J. Gen. Virol., 36:59-74 (1977)) or CHO cells. Theantibody protein was purified from cell-culture supernatants by proteinA affinity chromatography.

The LTα3 binding ELISA was carried out as follows: Microtiter wells werecoated with a 1 μg/ml LTα3 in 50 mM carbonate buffer solution (50μl/well) overnight. The unadsorbed solution was aspirated from thewells. Wells were blocked with 200 μL PBS containing 5 mg/ml bovineserum albumin (PBS-BSA) for 2 hours. 50 μL of test sample appropriatelydiluted in PBS-BSA was added to each well, incubated for one hour, andwashed with PBS containing 0.05% TWEEN-20™ surfactant. 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG in PBS-BSA buffer wasadded to each well and incubated for one hour. Each well was washed withPBS/0.05% TWEEN-20™ surfactant and then citrate phosphate buffer, pH 5,containing 0.1 mg o-phenylenediamine/ml (substrate solution), andaqueous 30% H₂O₂ was added to each well. The wells were incubated for 30minutes; then the reaction was stopped with 50 μL 2.5M sulfuric acidH₂SO₄, and absorbance was read at 490 nm.

The LTα3-blocking ELISA, the LTαβ-binding ELISA and FACS assays, and theLTαβ-blocking ELISA were carried out by procedures described furtherbelow.

EXAMPLE 2 Sequencing, Humanization, and Affinity Maturation of Anti-LTαAntibody 3F12

1. De Novo Sequencing of Anti-LTα 3F12 Using LTQ-FTMS-MS/MS Analysis andEdman Degradation

Because the hybridoma cell line 3F12.2D3 was lost from the frozen cellline bank, anti-LTα monoclonal antibody protein, purified from ascites3F12.2D3, was submitted for sequencing at a concentration of about 3.0mg/mL in PBS. The antibody (Ab) was separated on 4-20% TRIS™ HClSDS-PAGE under reducing conditions and each resolved heavy chain (HC)and light chain (LC) was subjected to N-terminal sequencing (25-30residues were sequenced) to establish monoclonality. The HC was found tobe blocked with a pyroglutamyl group that made the N-terminal amino acidinaccessible to Edman sequencing. The blocking group was subsequentlyremoved using the pyroglutamate aminopeptidase enzyme (PGAP) and the HCchain subjected to sequencing again. Both the HC and LC were confirmedto be monoclonal. The Ab was then treated with various enzymatic andchemical cleavages according to a strategy described in AnalyticalBiochemistry, 35:77-86 (2006). In brief, these consisted of the enzymestrypsin, endo Lys-C, Asp-N, and chemical treatments with CNBr and diluteacid (Asp/Pro cleavage).

Chemical cleavages generated a mixture of peptides that were sequencedon model 494 PROCISE™ sequencers and identified using Genentech'sSEQSORT program (SEQSORT is a utility program designed to make theoutput produced by the programs in Pittsburgh Supercomputing Center'ssequence analysis suite more manageable and readable). These chemicalcleavages allowed identification of the constant region of the HC and LCby identity with antibodies in protein sequence databases. Peptidesgenerated from the enzymatic digestions were collected from capillaryRP-HPLC separations and the peptide masses measured using MALDI-TOFanalysis. Peptides with masses that did not match the constant regionswere subjected to further analysis using Edman degradation or liquidchromatography-electrospray ionization tandem mass spectrometry(LC-MS/MS) on an LTQ-FT mass spectrometer. Sequences of the variableregions of the HC and LC were derived from peptides with overlappingsequences obtained by the various proteolytic cleavages.

For analysis of the peptides by LC-MS/MS a micro-capillary C18reverse-phase chromatography column (0.15×150 mm i.d., 5 μm, 300 Å) wasemployed using a flow rate of 1 mL/minute on an AGILENT™ 1100 seriescapillary LC system. Solvent A was applied, and a gradient from 2 to 80%solvent B was applied, solvent A consisting of 0.1% v/v formic acid inwater and solvent B consisting of 0.1% v/v formic acid in acetonitrile.The LC was coupled in-line with the LTQ-FT mass spectrometer, andpeptides with m/z between 350 and 1800 were analyzed in data-dependentmode with the five most abundant species in each full mass range surveyscan being selected for collision-induced dissociation (CID). Theresulting CID spectra were screened with the search program MASCOT tofind exact matches to database sequences, and potentially novelsequences were determined by manual de novo interpretation. Peptidesthat could not be completely sequenced or that contained the isobaricamino acid pairs of leucine/isoleucine and glutamine/lysine wereanalyzed further using Edman degradation.

A Fab portion of anti-LTα was obtained from papain digestion of theintact antibody followed by size-exclusion chromatography. Masses ofboth heavy-chain and light-chain components of the Fab were obtained byquadruple-TOF mass spectrometry after inter-chain disulfides werereduced. The experimental masses corresponded to those calculated basedon the obtained sequences of the heavy chain and light chain to within0.5 Da each.

Sequence of anti-LTα light chain for chimera 3F12.2D3:

(SEQ ID NO: 64) DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSTNQKNFLAWYQQKPGQSPKLLIYWASTRDSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSTSPIVKSFNRNECSequence of anti-LTα heavy-chain variable region spanning into constantregion for chimera 3F12.2D3:

(SEQ ID NO: 65) QGQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEISPGSGSTNYNEEFKGKATFTADKSSNTAYIQLSSLSTSEDSAVYYCADGYHGYWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVA HPASSTTVDKKLEPSGPISTwherein N is any amino acid.

FIG. 1C shows the light-chain variable region for this clone, as well asthe light-chain constant region as filled in by homology with otherantibodies, but not itself sequenced. FIG. 1D shows the heavy-chainvariable region for this clone, as well as the heavy-chain constantregion as filled in by homology with other antibodies.

2. Synthesis of DNA Encoding the Derived Amino Acid Sequence of Chimeric3F12

Using a pRK-based plasmid containing the gene for the light-chain ofanti-TGF-beta monoclonal antibody 2G7 (WO 2005/97832) as a template, andthe amino acid sequences derived above, several rounds ofoligonucleotide-directed mutagenesis were performed to derive the genefor the light chain of chimeric antibody 3F12. Similarly, the heavychain of monoclonal antibody 2G7 was the template for construction ofthe heavy chain of 3F12. Both light- and heavy-chain templates containedthe human constant domains, C kappa for the light chain, and CH1, hinge,CH2, and CH3 of IgG1 isotype, for the heavy chain. The oligonucleotidesused in the synthesis are given below for the light and heavy chains.

Oligonucleotides for Synthesis of Chimeric Light Chain of 3F12:

CA1845 (SEQ ID NO: 66) GCT CAT AGT GAC CTT TTC TCC AAC AGA CAC AGC CAGAGA TGA TGG CGA CTG TGA CAT CAC GAT ATC TGA ATG TAC TCC CA1846(SEQ ID NO: 67) GCT GTA AGT CCA GTC AAA GTC TTT TAT ACA GTA CCAATC AGA AGA ACT TCT TGG CCT GGT ACC AGC CA1847 (SEQ ID NO: 68)GCG ATC AGG GAC ACC AGA TTC CCT AGT GGA TGC CC CA1848 (SEQ ID NO: 69)GCC ACG TCT TCA GCT TTT ACA CTG CTG ATG G CA1849 (SEQ ID NO: 70)GGT CCC CCC TCC GAA CGT GCG CGG GTA GGA GTA GTATTG CTG ACA GTA ATA AAC TGC CAG GTCOligonucleotides for Synthesis of Chimeric Heavy Chain of 3F12:

CA1850 (SEQ ID NO: 71) GCT GCA GCT GAC CTT CTG AAT GTA CTC C CA1851(SEQ ID NO: 72) GCC TTG CAG GAG ATC TTC ACT GAA GCC CCA GGC TTCATC AGC TCA GCT CC CA1852 (SEQ ID NO: 73)CCC ACT CTA TCC AGT AAC TAG AGA AGG TGT ATC CAG TAG CCT TGC AGG CA1853(SEQ ID NO: 74) CCA CTT CCA GGA CTA ATC TCT CCA ATC CAC TCA AGGCCA TGT CCA GGC C CA1854 (SEQ ID NO: 75)GCC CTT GAA CTC CTC ATT GTA ATT AGT ACT ACC ACT TCC AGG CA1855(SEQ ID NO: 76) CCG CAG AGT CCT CAG ATG TGA TCA GGC TGC TGA GCTGGA TGT AGG CAG TGT TGG AGG ATT TGT CTG CAG TGA ATG TTG CCT TGC C CA1856(SEQ ID NO: 77) GGC TGA GGA GAC GGT GAC TGT GGT GCC TTG GCC CCAGTA GCC ATG GTA CCC GTC TGC ACA GTA ATA GAC CTC CA1871 (SEQ ID NO: 78)CCA GAC TGC TGC AGC TGA CCT TGT GAA TGT ACT CCA GTT GC

Since the original 3F12 murine monoclonal antibody had the isotype ofIgG2b, the murine constant domains for the light and heavy chains ofthis isotype were swapped for the human constant domains in the chimera,giving a fully murine antibody. For comparison, the murine IgG2a isotypewas also constructed. DNA for the light and heavy chain of each variantwas co-transfected into an adenovirus-transformed human embryonic kidneycell line, 293, for transient expression, and protein was purified fromconditioned media using Protein A affinity chromatography. Binding toLTα in an ELISA format was compared between the original 3F12 monoclonalantibody from ascites, and the synthetic mulgG2a and mulgG2b variants.

Briefly, a NUNC MAXISORP™ plate was coated with two micrograms per ml ofLTα (as described in Example 1 above) in 50 mM carbonate buffer, pH 9.6,overnight at 4° C., and then blocked with 0.5% BSA, 10 PPM PROCLIN™ inPBS at room temperature for 1 hour. Serial dilutions of samples in PBScontaining 0.5% BSA, 0.05% TWEEN™, 10 PPM PROCLIN™ were incubated on theplates for 2 hours. After washing, bound antibody was detected withhorseradish peroxidase-conjugated anti-human Fc (Jackson ImmunoResearch)using 3,3′,5,5′-tetramethyl benzidine (Kirkegaard & Perry Laboratories,Gaithersburg, Md.) as substrate. Absorbance was read at 450 nm. Both theIgG2a- and IgG2b-cloned variants bound similarly to the 3F12 antibodypurified from ascites, indicating that the correct sequence had beensynthesized.

3. Humanization of 3F12

Using the amino acid sequence of the chimeric clone, the CDR residues ofthe light and heavy chain were identified (Kabat).Oligonucleotide-directed mutagenesis was used to swap these CDRs ontovectors containing the coding sequences of the light and heavy chains ofhumanized anti-TGFβ 2G7 version 5 (WO 2005/97832), obtaining the CDRswapversion of 3F12. The framework sequences thus derived are for the VLkappa subgroup I and VH subgroup III consensus sequences, respectively:

(SEQ ID NO: 51) DIQMTQSPSSLSASVGDRVTITC- (SEQ ID NO: 55)L1-WYQQKPGKAPKLLIY- (SEQ ID NO: 53) L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC-(SEQ ID NO: 54) L3-FGQGTKVEIKR and (SEQ ID NO: 46)EVQLVESGGGLVQPGGSLRLSCAAS- (SEQ ID NO: 45) H1-WVRQAPGKGLEWVG-(SEQ ID NO: 50) H2-RATFSADNSKNTAYLQMNSLRAEDTAVYYCAD- (SEQ ID NO: 49)H3-WGQGTLVTVSS.

After expression and purification as described above, binding of theCDRswap to LTα was compared to that of the chimera. Binding for the CDRswap was almost completely lost.

To restore binding of the humanized antibody, mutations were constructedusing DNA from the CDRswap as template. Using a computer-generatedmodel, these mutations were designed to change human framework residuesto their murine counterparts at positions where the change might affectCDR conformation of the antibody-antigen surface. Mutants and theirrelative binding by ELISA as described above are shown in Table 1 below.Changing isoleucine 69 to phenylalanine, to give v2, increased bindingsignificantly, as did the change Leu78Ala, while the change Asn73Lysgave no improvement over the CDRswap. Combining Ile69Phe and Leu78Alagave version 5, which binds LTα approximately 5-fold less well than thechimera in this ELISA assay format.

Two variants were made to move the framework sequence closer to thehuman consensus. Version 6, with the changes Lys24Arg and Ser25Ala, wasequivalent in binding to version 5. However, an attempt to change theusual aspartic acid at position 94 to the consensus arginine (version 7)resulted in almost complete loss of binding.

TABLE 1 3F12 Heavy chain (VH) Light chain (VL) variant substitutionssubstitutions Relative binding* Chimera 1 CDRswap (CDR swap) (CDR swap)no binding v2 I69F (CDR swap) >1000x down v3 N73K (CDR swap) >1000x downv4 L78A (CDR swap) >500x down v5 I68F, L78A (CDR swap) 5 to 20 fold downv6 (CDR swap) K24R, S25A 5 to 20 fold down v7 D94R (CDR swap) no binding*These values are relative to the original chimera (with heavy chain SEQID NO: 35 and heavy-chain variable region set forth in FIG. 1B), whichwas discovered later to have an “extra” serine in the FR3 of the heavychain (a serine after the L at position c in SEQ ID NO: 29 in FIG. 3B).This serine was taken out to make chimera2 (having the heavy-chainvariable region of SEQ ID NO: 38 in FIG. 1D), and the ELISA binding wasfound to be the same as above.

All animal studies described below using the 3F12 chimera refer to theoriginal chimera, and not chimera2 as described in the footnote above.

4. Affinity Maturation of Antibody 3F12

For identification of which residues and regions in the CDRs might bemost important for binding LTα, and thus candidates for inclusion inphage-display libraries for affinity maturation, partial alaninescanning was performed, using v5 as single-strand template foroligonucleotide-directed mutagenesis.

As shown in Table 2, several residues were found which when mutated toalanine produced a loss or gain in binding to LTβ.

TABLE 2 version # Position relative binding* L1 13 Y31A 3.40 14 T33A0.72 15 N34A 0.49 16 K36A 3.70 17 F38A 0.32 L3 18 Q89A 4.20 19 Y91A 2.3020 Y92A 0.71 21 S93A 0.82 22 Y94A 14.30 23 R96A 18.20 H2 24 G53A 34.1025 G55A 1.20 26 S56A 1.07 27 N58A 1.70 28 Y59A 2.73 29 N60A 2.31 30 E61A1.78 31 E62A 1.93 H3  8 G95A >100  9 Y99A 10.00 10 H100A >100 11 G101A12 Y102A 0.81 *The ratio of the EC50 of the variant divided by the EC50of version 5 in plate-based ELISA against LTα. Higher numbers meanweaker binding, numbers >1 mean binding is less than that of version 5.

Thus, versions 14, 15, 17, 20, 21, and 12 have positions where changescould be made to improve binding affinity, and it is expected thatconservative substitutions (or even non-conservative in some instancesas determined by the skilled artisan by routine testing) could be madeat those respective positions to increase binding.

EXAMPLE 3 Preparation of Chimeric Antibody 2C8 and Humanization

1. Preparation of Chimeric Antibody

The variable regions of the heavy and light chains of the murineanti-LTα antibody 2C8 that bound and neutralized LTα3 and LTαβ werecloned and formatted as a murine/human chimeric antibody essentially asdescribed above in Example 1 for the cloning of S5H3.

For RT-PCR, the primers used were as follows:

2C8 light chain: (SEQ ID NO: 79) 5′primer: GCC ATA GAT ATC GTR ATG CAN CAG TCT C (SEQ ID NO: 80) 3′primer: TTT KAT YTC CAG CTT GGT ACC 2C8 heavy chain: (SEQ ID NO: 81) 5′primer: GAT CGA CGT ACG CTG AGG TYC AGC TSC AGC TSC AGC AGT CTG G(SEQ ID NO: 82) 3′ primer: ACA GAT GGG CCC TTG GTG GAG GCT GMR GAGACD GTG ASH RDR GT.wherein K=G or T, S=C or G, D=A or G or T, M=A or C, H=A or C or T, N isT, G, A, or C (any nucleic acid of these four), R=A or G, and Y=C or T.

The variable domain of the light chain was cloned into vectorpRK.LPG3.HumanKappa containing the human kappa constant domain, whilethe heavy-chain variable domain was cloned into expression vectorpRK.LPG4.HumanHC encoding the full-length human IgG1 constant domains.These vectors are derivatives of previously described vectors forexpression of IgGs in 293 cells (Shields et al., J Biol Chem,276:6591-6604 (2000) and Gorman et al., DNA Prot Eng Tech, 2:3-10(1990)). For transient expression of antibody, these plasmids wereco-transfected into 293 cells or CHO mammalian cell lines and theprotein was purified as described in Example 1 above.

This chimeric antibody 2C8 was used in the animal experiments describedbelow.

2. Humanization of 2C8

Humanization of the anti-LTα antibody 2C8 was carried out in a series ofsite-directed mutagenesis steps. Using the sequence of the variabledomains of the chimera, the CDR residues of the light and heavy chainswere identified by comparing the amino acid sequence of these domainswith the sequences of known antibodies (Kabat et al., supra). The CDRswere defined based on sequence hypervariability (Kabat et al., supra)and are shown in FIG. 2.

The CDR swap of 2C8 was made using oligonucleotide site-directedmutagenesis, wherein the CDR regions were put onto separate pRK5-basedvectors for the light and heavy chains, respectively. These vectorscontain the light-chain human kappa I constant domain, or for theheavy-chain vector, the human IgG1 constant domains CH1, hinge, CH2, andCH3 (Gorman, Current Opinion in Biotechnology, 1, (1), p 36-47 (1990)).

Comparison to other humanized antibodies indicated that the light chainof 2C8 is very similar to the light chain of humanized anti-HER2antibody 4D5 (U.S. Pat. No. 5,821,337), while the heavy chain of 2C8 issimilar in sequence to anti-TGF-β 2G7 (WO 2005/97832). Therefore, avector containing the light chain of rhuMAb 4D5 was used as the templatefor the construction of the light chain of 2C8. Site-directedmutagenesis was performed on a deoxyuridine-containing template derivedfrom this vector, using oligonucleotides with light-chain sequencesshown in Table 3. Additionally, the oligonucleotide CA1730 was used torevert the framework 3 sequence to that of the human kappa I consensus.

TABLE 3 Oligonucleotides for 2C8 (light chain) SubstitutionOligonucleotide sequence CDR-L1 CC TGG TTT CTG TTG ATA CCA GGC TACAGC GGA AGA CAC AGC CTG ACT GGC ACG GCA GG (SEQ ID NO: 83) CDR-L2GCG AGA AGG GAC TCC AGT GTA ACG GTG GGA TGC AGA GTA AAT CTG TAG TTT CGGAGC (SEQ ID NO: 84) CDR-L3 GGT ACC CTG TCC GAA CGT CCA AGG AGTAGA ATA ATG TTG CTG ACA GTA ATA AGT TGC (SEQ ID NO: 85) R66G (CA1730)GGT CAG AGT GAA ATC CGT CCC AGA TCC GGA TCC AGA GAA GCG (SEQ ID NO: 86)(All the oligonucleotides were ordered as reverse complements to thecoding strand, as the f1 ori in pRK vectors is in the oppositeorientation from that in pBR-based vectors.)

For the heavy chain, the deoxyuridine-containing template was derivedfrom a vector containing the heavy chain of humanized antibody 2G7 (WO2005/97832) using oligonucleotides shown in Table 4. The framework wasconverted to consensus using oligonucleotides KM55, KM56, and KM61.

TABLE 4 Oligonucleotides for 2C8 (heavy chain) CDR-H1GGC CTG ACG GAC CCA ATG GAT CAC ATA GCT GGT GAA TCT GTA GCC AGA AGC TGC(SEQ ID NO: 87) CDR-H2 CCC CTT GAA CTT CTC GTT ATA GTT GGT GCCGTC GTT ATA AGG ATT GTT ATA ACC AAC CCA CTC GAG GCC (SEQ ID NO: 88)CDR-H3 GGT TCC TTG ACC CCA GTA GGC GAA CCA TGGGAG CAT TGT GGG TCG AGA ACA ATA ATA GAC GGC AGT GTC CTC AGC(SEQ ID NO: 89) KM55 CC GTC GTT ATA AGG ATT GTT ATA ACT AACCCA CTC GAG GCC (SEQ ID NO: 90) KM56CAT CTG CAG GTA TAG TGT GTT TTT CGA ATTGTC ACG ACT GAT AGT GAA GCG CCC CTT GAA C (SEQ ID NO: 91) KM61CCA TGG GAG CAT TGT GGG TCG AGC ACA ATA ATA GAC GGC AGT GTC C(SEQ ID NO: 92)

The light and heavy chains comprising the CDR swap, v10, thus derived,were co-transfected into an adenovirus-transformed human embryonickidney cell line, 293 (Graham et al., supra). Antibodies were purifiedfrom culture supernatants using protein A-SEPHAROSE CL-4B™ resin, thenbuffer-exchanged into 10 mM sodium succinate, 140 mM NaCl, pH 6.0, andconcentrated using a CENTRICON-10™ concentrator (Amicon).

For measuring relative binding affinities of the CDR swap, andsubsequent 2C8 variants, to human LTα, a plate-based ELISA was used asdescribed above.

As shown in Table 5, the CDR swap, v10, shows no binding in the LTαELISA. Version 2 and subsequent versions were constructed to regain thisbinding. Version 2 regains binding equivalent to the chimera, butcontains seven non-consensus residues. Further variants were studied toobtain a framework as close to consensus as possible while stillretaining or improving binding relative to the chimera. Version 12 hasbinding equivalent to (by ELISA), or slightly better than (by BIACORE™analysis), the chimera, and retains only five non-consensus residues.The light- and heavy-chain sequences of versions 7 and 12 are providedin FIG. 2C.

TABLE 5 ELISA IC₅₀ BIACORE ™ (rhLTα) 2C8 version HC LC ratio KD ka/kdmutations v0 v0 v0 1 0.6 nM v2 v2 v1 1.7 0.2 nM 6e5/1e−4 v3 v3 v1 10.2v4 v4 v1 27.8 v7 v2 v2 1.2 0.4 nM 5e5/1.6e−4 LC.Q46L v8 v8 v1non-binding HC.G49S.S93A v10 v10 v1 non-binding CDRswap v12 v12 v2 10.07 nM  2e6/0.9e−4 HC.A67F v13 v13 v2 2.4 HC.S71R v14 v14 v2 2 HC.K73Nv15 v15 v2 11.1 HC.A93S v16 v12 v3 0.6 LC.Q46L.E89Q.S90H vX vX v3 0.3HC.D53A.A67F3. Affinity Maturation of 2C8

A humanized 2C8 monoclonal antibody with improved affinity to LTα wasselected from a library of monovalent Fab displayed on phage. A templatewas generated that displays the humanized 2C8.v2 Fab and contains a stopcodon in the CDR-L3. Oligonucleotides (listed in Table 6) were designedto soft-randomize specific CDR-L3 and CDR-H3 residues.

TABLE 6 Soft randomization oligonucleotides for affinitymaturation of 2C8 CDR-L3 and CDR-H3 Oligonucleotide name Sequence¹KM73.2C8.phage. GGA AGA CTT CGC AAC TTA TTA CTG v3.LC3.softTCA G75 575 885 887 857 8CC T86 6AC GTT CGG ACA GGG TAC CAA GGT GG(SEQ ID NO: 93) KM74.2C8.phage. G GAC ACT GCC GTC TAT TAT TGTv3.HC3.soft TCT CGA CCC 575 576 787 CCA 866887 677 TAC TGG GGT CAA GGA ACC CTG G (SEQ ID NO: 94) ¹Softrandomization code (A = 5, G = 6, C = 7 and T = 8): each degenerate baseis 70% wild-type with 10% equimolar amounts of the remaining threenucleotides added.

Three rounds of sorting on LTα were used to selectively capturehigh-affinity Fab molecules with less than 2 nM binding to the antigen.For the first round, phage were captured for 2 hours against 2 μg/mlplate-coated LTα at ambient temperature. The second and third rounds ofselection were performed at ambient temperature by capturing phage for30 minutes with 2 nM biotinylated LTα in solution phase, followed bycapture on neutravidin-coated plates. The highest affinity clone wasidentified using a competitive ELISA. Constant concentrations of phageclones (normalized by solid-phase LTα binding OD) were incubated withincreasing concentrations of LTα (0-50 nM) in solution before capture ona plate coated with 2 μg/ml LTα. One clone demonstrated a 10-foldimprovement in competition with LTα over wild-type 2C8.v2. DNAsequencing of this phagemid clone revealed two amino acid changes fromthe wild-type 2C8 sequence in CDR-L3: glutamine at position 89 waschanged to a glutamic acid, and histidine at position 90 was changed toa serine. These two CDR-L3 changes were made by site-directedmutagenesis on the wild-type 2C8.v7 light chain. When v7 light chain wastransfected into 293 cells with the 2C8.v12 heavy chain to give 2C8.v16,the resultant antibody demonstrated a two-fold improvement in IgGbinding to LTα by ELISA when compared to 2C8.v2 (Table 5).

To enhance stability, by removing a potential aspartic acidisomerization site, asp 53 in heavy-chain CDR2 was changed to alanine.The resultant 2C8 variant, vX, had a 3.5-fold and 3-fold improvement inbinding to LTα by ELISA, compared to the 2C8.v2 and 2C8.chimera,respectively (Table 5).

Table 7 below shows how residue changes in CDR-L3 regions ranked as toability to bind LTα by ELISA analysis.

TABLE 7 Affinity maturation of 2C8 by phage display: sequence changes in phagemid CDR-L3 regionsranked by ability to compete rhLTα Phagemid rhLTα Competitive cloneCDR-L3 Sequence^(1,2) ELISA IC₅₀ (nM) Wild-type  Q

T  3.14 (SEQ ID NO: 9) A8 Q

T  0.32 (SEQ ID NO: 10) G7 Q

T  0.57 (SEQ ID NO: 11) H6 Q

T  1.1 (SEQ ID NO: 12) C12 Q

T  3.41 (SEQ ID NO: 95) H9 Q

T  3.80 (SEQ ID NO: 96) G2 Q

T  4.51 (SEQ ID NO: 97) G12 Q

T  5.34 (SEQ ID NO: 98) D8 Q

T  136.62 (SEQ ID NO: 99) E2 Q

T  not determined³ (SEQ ID NO: 100) ¹Boldface and italicized type showsresidues randomized. ²All phagemids retained the wild-type CDR-H3sequence ³Clone E2 did not meet rhLTα direct ELISA binding threshold (ofOD = 1) necessary for phagemid normalization in competitive ELISA.

It can be seen from this table that the best clones were A8, G7, and H6,which all bound rhLTα with higher affinity than the wild-type clone.

EXAMPLE 4 Anti-LTα Antibody Inhibits Antibody-Induced Arthritis (AIA)

AIA differs from collagen-induced arthritis (CIA), described below, inthat instead of injecting the antigen (bovine collagen type II),antibodies recognizing type II collagen are injected. In this way,adaptive B- and T-cell responses are circumvented to directly induceeffector functions on macrophages and neutrophils through Fc receptorand complement-mediated activation.

AIA was induced in mice by i.v. injection of a combination of fourdifferent monoclonal antibodies generated by the ARTHROGEN-CIA™ mouseB-hybridoma cell lines. Three of the monoclonal antibodies recognizeautoantigenic epitopes clustered within an 84-amino-acid residuefragment, LyC2 (the smallest arthritogenic fragment of type II collagen)of CB11, and the fourth monoclonal antibody reacts with LyC1. All fourantibodies recognize the conserved epitopes shared by various species oftype II collagen and crossreact with homologous and heterologous type IIcollagen.

All groups (n=5/group) received 2 mg of a cocktail of monoclonalantibodies i.v. in 100 μl PBS on day 0 followed by 25 μg LPS i.p. in 50μl PBS on day 3. Animals were treated on Day −1 (preventive) or Day 4(therapeutic) with 10 mg/kg control isotype (anti-ragweed),hamster-murine chimeric LTα antibody S5H3 (as a surrogate for theanti-human LTα antibodies), anti-LT.Fc mutant (an anti-LTα antibody withDANA mutations in the Fc region as described below), or TNFRII.Fc (aTNFR.Ig immunoadhesin) intraperitoneally or subcutaneously. Swelling ofthe paws was monitored for 14 days. Each paw was given a score of 0-4,for a total maximum of 16 per animal.

DANA mutations in the anti-LT.Fc mutant used in this Example and othersbelow refer to two single-position changes in the Fc region of mouse orhuman IgG. These changes are D (Aspartic Acid) to A (Alanine) inposition 265 and N (Asparagine) to A (Alanine) in position 297. The DANAmutant was constructed by introducing alanine residues into positions265 and 297 of mouse or human IgG by a PCR-based site-directedmutagenesis method (GENE TAILOR™, Invitrogen Corp.).

FIG. 4A shows the average clinical score for the prophylactic treatmentof each group described above as a function of days post-induction. Itcan be seen that the S5H3 antibody was the most effective in this modelof all the treatment groups, including the TNFRII.Fc and anti-LT.Fcmutant molecules. Thus, antibody S5H3 significantly inhibited thedevelopment of arthritis in this antibody-induced model. Anti-LTα withthe Fc DANA mutation (anti-LT.Fc mutant) had no efficacy, indicatingthat Fc-mediated killing of LTβ-expressing cells was required forefficacy (FIG. 4A).

FIG. 4B shows the average clinical score for therapeutic treatment ofeach group described above as a function of days post-induction. Theresults show that the S5H3 antibody significantly inhibited thedevelopment of arthritis in this antibody-induced model whenadministered therapeutically, versus the other molecules tested.

In conclusion, animals treated with the hamster-mouse chimera S5H3 hadsignificantly reduced clinical scores as compared to animals treatedwith S5H3.DANA or control. S5H3 demonstrated both prophylactic andtherapeutic efficacy in this animal model.

EXAMPLE 5 Anti-LTα Inhibits Collagen-Induced Arthritis (CIA)

In RA the synovial membrane of multiple joints can become inflamed,leading to destruction of joint tissues including bone and cartilage.The synovium of RA can be highly inflammatory in nature and is typicallycharacterized by lymphocyte and mononuclear cell infiltration, T-celland antigen-pressing cell (APC) activation, B-cell immunoglobulin (Ig)secretion, and pro-inflammatory cytokine production (Potocnik et al.,Scand. J. Immunol., 31:213 (1990); Wernick et al., Arthritis Rheum.,28:742 (1985); Ridley et al., Br. J. Rheumatology, 29:84 (1990); Thomaset al., J. Immunol., 152:2613 (1994); and Thomas et al., J. Immunol.,156:3074 (1996)). Chronically inflamed synovium is usually accompaniedby a massive CD4 T-cell infiltration (Pitzalis et al., Eur. J. Immunol.,18:1397 (1988) and Morimoto et al., Am. J. Med., 84:817 (1988)).

Collagen-induced arthritis (CIA) is an animal model for human RA, whichresembles human disease, and can be induced in susceptible strains ofmice by immunization with heterologous type-II collagen (CII) (Courtenayet al., Nature, 283:665 (1980) and Cathcart et al., Lab. Invest., 54:26(1986)). Both CD4 T cells and antibodies to CII are required to developCIA. Transfer of anti-CII to naïve animals only leads to partialhisto-pathology that is quite different from CIA, and complete symptomsof CIA do not develop (Holmdahl et al., Agents Action, 19:295 (1986)).In contrast, adoptive transfer of both CD4 T cells and anti-CIIantibodies from CII-immunized mice to naïve recipients completelyreconstitutes the symptoms of classical CIA (Seki et al., J. Immunol.,148:3093 (1992)). Involvement of both T cells and antibodies in CIA isalso consistent with histo-pathological findings of inflamed joints inCIA. Thus, agents that block B-cell or T-cell functions, or inhibitpro-inflammatory cytokines induced by T cells, may be efficacious toprevent or treat arthritis. Indeed, depletion of CD4 T cells, blockadeof CD40-CD40L interactions, neutralization of TNF-α, or blocking of IL-1receptors can lead to prevention of CIA in mice (Maini et al., Immunol.Rev., 144:195 (1995); Joosten et al., Arthritis Rheum., 39:797 (1996);and Durie et al., Science, 261:1328 (1993)).

In the CIA model used herein, DBA-1J mice were immunized with 100 μgbovine collagen type II in 100 μl of Complete Freund's Adjuvant (CFA) onDay 0 and Day 21 intradermally. At Day 24 post-immunization, mice wererandomly divided into treatment groups. Animals were subcutaneouslytreated either with 6 mg/kg in 100 μl anti-ragweed IgG2a monoclonalantibody (control antibody) or with hamster-mouse chimeric anti-LTαantibody (S5H3), anti-LTα.Fc mutant, or murine TNFRII-Ig at 4 mg/kg in100 PBS. Animals were treated three times weekly for the duration of thestudy. Limbs of animals were examined daily for signs of jointinfiltration using a grading system of 1-4 for each joint, giving amaximum score of 16.

In a CIA-preventative model, the use of hamster-murine anti-LTα antibodyS5H3 and TNFRII.Fc reduced the average clinical score over the period ofdays up through 70 days versus the anti-S5H3.DANA Fc mutant and isotypecontrol (anti-ragweed IgG2a monoclonal antibody). See FIG. 5A, showingcomparable efficacy in the CIA preventative murine model of the antibodyS5H3 with TNFR.Ig.

FIG. 5B shows comparable efficacy of the hamster-murine chimericanti-LTα antibodies (S5H3) with TNFRII.Fc over 90 days in thistherapeutic CIA murine model, which efficacy is far better than theisotype control in reducing average clinical score. These results alongwith those in FIG. 5A show that the antibodies herein are considereduseful in preventing and being efficacious in autoimmune diseases suchas RA. It is expected that the anti-LTα antibodies herein would beeffective in preventing and treating RA in those who are non-responsiveto TNF therapies in general, including TNFR.Ig and anti-TNFα antibodies,so that such non-responders could be treated with such antibodiesprophylactically and efficaciously.

EXAMPLE 6 Anti-LTα Delays Onset and Severity of Encephalomyelitis (EAE)Disease in MBP-TCR Transgenic Mice

Experimental autoimmune/allergic encephalomyelitis (EAE) is aninflammatory condition of the central nervous system with similaritiesto MS. In both diseases, circulating leukocytes penetrate theblood-brain barrier and damage myelin, resulting in impaired nerveconduction and paralysis. The EAE murine model (transgenic mousepreventative model) has been described as a model for human MS (Grewalet al., Science, 273:1864-1867 (1996)).

MBP_(Acl-11) T-cell receptor transgenic mice (10-to-15 week-old male andfemale adult MBP-TCR transgenic mice bred from an animal breeding pairobtained from Dr. Richard Flavell, Howard Hughes Medical Institute, YaleUniversity) were immunized with 10 μg of MBP_(Acl-11) in 100 μl of CFAsubcutaneously. On Days 1 and 2 all mice were additionally injectedintraperitoneally with pertussis toxin at 200 ng/10 μl/mouse. On Day 0,mice (n=10/group) received either anti-gp120 IgG1 monoclonal antibody(control antibody) or hamster-mouse chimera anti-LTα antibody S5H3 at 6mg/kg in 100 μl i.p. and then 6 mg/kg in 100 μl PBS s.c. three timesweekly.

Animals were evaluated daily for clinical signs using the followinggrading system: 0—Normal mouse; no overt signs of disease; 1—Limp-tailor hind-limb weakness, but not both; 2—Limp-tail and hind-limb weakness;3—Partial hind-limb paralysis; 4—Complete hind-limb paralysis;5—Moribund.

The results are shown in FIG. 6. The disease score was lower for theanti-LTα-treated mice than for the control group, only reaching clinicalscores of 3 (versus clinical scores of over 4 for the control) duringthe study. The results show that the antibody S5H3 delayed onset andseverity of EAE disease in these transgenic mice, suggesting that theantibody treatment protected the mice from developing overt EAE.

EXAMPLE 7 Anti-LTα Antibody Treatment Does Not Affect T-Cell-DependentAntibody Isotype Responses, Indicating Safety

The immune response to i.v.-injected trinitrophenol (TNP)-FICOLL™ isvery high in most mouse strains, allowing one to assay the relativesafety of an antibody administered to such mice by measuring variousisotype levels in the mice.

BALB/c mice (n=12/group) were treated on Day 0 with either anti-ragweedisotype control, hamster-mouse anti-LTα antibody (S5H3), anti-LT.Fcmutant, or CTLA4-Fc (as a positive control) at 6 mg/kg in 100 μl PBSintra-peritoneally. Treatment continued three times per week for 5weeks. Mice were immunized on Day 1 with TNP-OVA (100 μg) in 2 mg ofalum per mouse intra-peritoneally, and on Day 29 with TNP-OVA (50 μg) inPBS per mouse intra-peritoneally. Serum was collected on Days 0, 14, and35 for determination of the anti-TNP Ig isotypes IgM, IgG1, and IgG2a bystandard ELISA.

The results, shown in FIGS. 7A-7C, wherein the data are represented asLog 10 titers of anti-TNP IgM, IgG1, and IgG2a, respectively, indicatethat the isotype control, S5H3 antibody, and DANA Fc mutant antibodyexhibited similar patterns for each of the isotype levels, whereas theCTLA4.Fc immunoadhesin acted differently. This indicates that therewould be little, if any, immune response in a patient to theadministration of the S5H3 antibody herein.

EXAMPLE 8 Anti-LTα Antibody Treatment Does Not Affect T-Cell-IndependentAntibody Isotype Responses, Indicating Safety

BALB/c mice (n=10/group) were treated on Day 0 with either anti-ragweed(IgG2a isotype control), hamster-mouse anti-LTα antibody (S5H3), orTNFRII.Fc at 6 mg/kg in 100 μl PBS intra-peritoneally. Treatmentcontinued three times per week for 10 days. Mice were immunized on Day 1with TNP-FICOLL™ (100 μg) i.p. in 100 μl PBS intra-peritoneally. Serumwas collected on Days 0 and 10 for determination of the anti-TNP Igisotypes IgM, IgG1, IgG2a, and IgG3 by standard ELISA.

FIG. 8, wherein the data are represented as Log 10 titers, shows thatthe isotype control, the S2C8 antibody, and the TNFRII.Fc immunoadhesinall behaved similarly for the IgM, IgG1, IgG2a, and IgG3 isotypes. Thisdemonstrates further the safety of the antibodies herein foradministration in vivo.

EXAMPLE 9 Anti-LTα Antibody Prevents Human SCID Graft-Versus-HostDisease (GVHD)

Graft-versus-host disease (GVHD) occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor Tcells recognize host antigens and become activated, secrete cytokines,proliferate, and differentiate into effector cells. This response isknown as graft-versus-host-reaction (GVHR). The GVHR response is amulti-organ syndrome, and the effects can vary from life-threateningsevere inflammation to mild cases of diarrhea and weight loss. GVHDmodels in mice have been used to model the clinical disorders of acuteand chronic GVHR that occur after bone-marrow transplantation andautoimmune diseases. A general procedure is described in CurrentProtocols in Immunology, supra, unit 4.3.

SCID mice were reconstituted with human PBMCs purified from a LEUKOPACK™(available from blood banks such as Interstate Blood Bank, Memphis,Tenn.) of a normal donor by FICOLL™ polysaccharide gradient. All mice(n=10/group) were sub-lethally irradiated with 350 rads using a Cesium137 source. Two hours after irradiation, mice were injected with 50million human PBMCs/mouse in 200 μl PBS intravenously. Immediately aftercell injection, mice were treated i.p. either with 300 μg of trastuzumab(human IgG1 isotype control antibody), anti-LTα chimeric antibody 2C8,or CTLA4-Fc in 100 μl saline two times/week for three weeks. POLYMYXIN™B (110 mg/liter) and NEOMYCIN™ (1.1 g/liter) antibiotics were added tothe drinking water for five days post-irradiation. Mice were monitoredfor GVHD as indicated by survival.

The results are shown in FIG. 9, and indicate that as compared to theisotype control, the 2C8 antibody significantly increased survival ofthe human SCID mice (preventing GVHD), as compared to anti-LTα 2C8 Fcmutant and the isotype control, with the CTLA4.Fc immunoadhesin beingthe best in this model of the molecules tested. Thus, the anti-LTαantibodies herein are expected to be useful in the prevention and/ortreatment of graft-versus-host disease. Further, the results show thatdepletion of LTβ-positive cells is required for efficacy in this model,since the 2C8 Fc mutant (DANA) did not show efficacy.

EXAMPLE 10 Anti-LTα Monoclonal Antibodies Bind to LTα3

Microtiter wells were coated with 1 μg/ml human LTα3 in 50 mM carbonatebuffer solution (100 μl/well) overnight. The unabsorbed solution wasdecanted from the wells. Wells were blocked with 150 μL PBS containing 5mg/ml bovine serum albumin (PBS-BSA) for 1-2 hour. 100 μL ofappropriately diluted test sample (anti-LTα chimeric antibody 2C8 or3F12) diluted in PBS-BSA was added to each well, incubated for one hour,and washed with PBS containing 0.05% TWEEN™-20 surfactant. 100 μL ofbiotin-labeled rat anti-mouse IgG in PBS-BSA buffer was then added toeach well and incubated for one hour. The plate was then washed withPBS/0.05% TWEEN™-20 surfactant, and streptavidin-horseradish peroxidase(SA-HRP) was added for 30 minutes to each well. Each well was washedwith PBS/0.05% TWEEN™-20 surfactant, and bound HRP was measured with asolution of tetramethylbenzidine (TMB)/H₂O₂. After 15 minutes, thereaction was quenched by the addition of 100 μl of 1M phosphoric acid.The absorbance at 450 nm was read with a reference wavelength of 650 nm.

FIG. 10A shows that chimeric anti-LTα antibodies 2C8 and 3F12 bound tohuman LTα3.

The above procedure was repeated using murine LTα3 instead of human LTα3and hamster-mouse chimeric anti-LTα antibody S5H3 and anti-LT.Fc mutantas the antibody tested for binding ability. FIG. 10B shows that bothS5H3 and the anti-LTα.Fc mutant bound to murine LTα3.

EXAMPLE 11 Anti-LTα Antibodies Bind to LTα1β2

For the LTαβ ELISA, microtiter wells were coated with 1 μg/ml murineLTα1β2 in 50 mM carbonate buffer solution (100 μl /well) overnight. Theunabsorbed solution was decanted from the wells. Wells were blocked with150 μL PBS containing 5 mg/ml bovine serum albumin (PBS-BSA) for 1-2hours. 100 μL of an appropriately diluted test sample (anti-LTα chimericantibody S5H3 or anti-LTα.Fc mutant diluted in PBS-BSA) was added toeach well, incubated for one hour, and washed with PBS containing 0.05%TWEEN™ 20 surfactant. 100 μL of biotin-labeled rat anti-mouse IgG inPBS-BSA buffer was then added to each well and incubated for one hour.The plate was then washed with PBS/0.05% TWEEN™ 20 surfactant, andstreptavidin-horseradish peroxidase (SA-HRP) was added for 30 minutes toeach well. Each well was washed with PBS/0.05% TWEEN™ 20 surfactant, andbound HRP was measured with a solution of tetramethylbenzidine(TMB)/H₂O₂. After 15 minutes, the reaction was quenched by the additionof 100 μl of 1M phosphoric acid. The absorbance at 450 nm was read witha reference wavelength of 650 mm.

FIG. 11A shows that anti-LTα chimeric antibody S5H3 bound to the murineLTα1β2 complex, as did the anti-LT.Fc mutant.

A FACS assay was used to determine binding of anti-LTα antibody to theLTα on the surface of cells complexed with LTβ. For human LTαβ, 293cells were transfected with full-length human LTα and human LTαβ (humanLTα sequence GenBank Ref NM_(—)000595; human LTβ sequence GenBank RefNM_(—)002341) to generate stable human LTαβ-expressing cell lines. Cellswere incubated with 1-5 μg/ml of anti-LTα chimeric antibody 3F12 or 2C8,human LTβR.huIgG1, or huIgG1 isotype control (trastuzumab) for 20minutes in PBS with 2% FBS. Cells were washed and incubated withanti-human Ig-PE secondary antibodies for detection.

For murine LTαβ, SVT2 cells were transfected with full-length murine LTαand murine LTαβ (murine LTα sequence GenBank Ref NM_(—)010735; murineLTβ sequence GenBank Ref NM_(—)008518) to generate stable murineLTββ-expressing cell lines. Surface LT was detected with hamster-murinechimera S5H3, anti-LT.S5H3.Fc mutant, muLTβR.IgG2a, or isotype control(anti-IL122 mulgG2a) directly conjugated to ALEXA-647™ fluorophore, for20 minutes in PBS with 2% FBS. Cells were washed, aspirated, andresuspended in PBS with 2% FBS. Cells were analyzed on a FACSCALIBUR™(dual laser) using CELLQUEST™ software, and results analyzed on aFLOWJO™ analyzer (Treestar).

FIG. 11B shows how well the chimeric anti-LTα antibodies 3F12 and 2C8bound to the LTα on the surface of human cells complexed with LTβ. Theyare compared to LTβ-receptor.huIgG1 and isotype control huIgG1(trastuzumab). All human antibodies bound surface LTα.

FIG. 11C shows how well hamster-murine anti-LTα antibody S5H3 bound tothe LTα on the surface of murine cells complexed with LTβ. It iscompared to LTβ-receptor murine IgG2a, anti-LT S5H₃Fc mutant, andisotype control (anti-IL122 mulgG2a). All murine antibodies boundsurface LTα.

EXAMPLE 12 Anti-LTα Antibodies Bind to Human Th1, Th2, and Th17 Cells

When CD4+ T cells mature from thymus and enter into the peripheral lymphsystem, they usually maintain their naive phenotype before encounteringantigens specific for their T cell receptor (Sprent et al., Annu RevImmunol. 20:551-79 (2002)). The binding to specific antigens presentedby APC, causes T cell activation. Depending on the environment andcytokine stimulation, CD4+ T cells differentiate into a Th1 or Th2phenotype and become effector or memory cells (Sprent et al., supra andMurphy et al., Nat Rev Immunol. 2(12):933-44 (2000)). This process isknown as primary activation. Having undergone primary activation, CD4+ Tcells become effector or memory cells, they maintain their phenotype asTh1 or Th2. Once these cells encounter antigen again, they undergosecondary activation, but this time the response to antigen will bequicker than the primary activation and results in the production ofeffector cytokines as determined by the primary activation (Sprent etal., supra, and Murphy et al., supra).

For primary activation conditions, naïve T cells were activated byanti-CD3, anti-CD28, and specific cytokines depending on whether Th1 orTh2 was being examined. In particular, human Th1 and Th2 cell lines weregenerated by stimulating human PBMC with plate-bound anti-CD3 andanti-CD28 (10 μg/ml and 5 μg/ml, respectively; BD PharMingen). On Day 1,to induce Th2 differentiation, interleukin (IL)-4 (5 ng/ml; R&D SystemsInc.), anti-IL-12 (5 μg/ml; R&D Systems Inc., Minneapolis, Minn.), andanti-interferon (IFN)-gamma (5 μg/ml; R&D Systems Inc.) were added. ForTh1 differentiation, on Day 1 IL-12 (1 ng/ml; R&D Systems Inc.),IFN-gamma (10 ng/ml; R&D Systems Inc.), and anti-IL-4 (1 μg/ml; R&DSystems Inc.) were added. Cells were restimulated twice at Days 7 and14.

For FACS analysis cell-surface staining to determine binding of theantibodies to Th1 and Th2, two days after final stimulation, theactivated human T-cells (Th1/Th2 cell lines) were incubated withanti-LTα antibody 2C8 (1 μg/ml), 3F12 (1 μg/ml), LTβR.Fc (1 μg/ml),TNFRII.Fc (1 μg/ml) or huIgG1 isotype control (1 μg/ml), all directlyconjugated to ALEXA FLUOR® 647 fluorophore (Invitrogen Corp.), for 20minutes in PBS with 2% FBS. Cells were washed, aspirated, andre-suspended in PBS with 2% FBS. Cells were analyzed on a FACSCALIBUR™(dual laser) using CELLQUEST™ software, and results analyzed on aFLOWJO™ analyzer (Treestar).

FIGS. 12A and 12B show that chimeric anti-LTα antibodies 2C8 and 3F12bound to human Th1 and Th2 cells, respectively. The graphs also show thebinding results for LTβR.Fc, TNFRII.Fc, and isotype control.

Since LT-α3 and LT-α1β2 were found to be expressed on human Th17 cellsas well as human Th1 cells via FACS analysis, it would be expected thatthe anti-LTα antibodies herein, including chimeric 2C8, 2C8.v2, and2C8.vX (described in Examples 3 and 18), would (similarly to Th1 andTh2) bind to human Th17 cells and hence be useful in treating manyautoimmune diseases, including MS (they drive EAE in murine models) andlupus as well as RA and IBD, such as Crohn's disease and ulcerativecolitis.

Hence, the same experiment described above was performed, except forusing anti-LTα humanized antibody 2C8.vX as the LTα antibody and using aTh17 cell line as well as the Th1 cell line, but not a Th2 cell line.The human Th17 cell lines were generated by stimulating human PBMC withanti-CD3 and anti-CD28 as noted above and with IL-23 (10 ng/ml; R&DSystems Inc.), anti-IFN-gamma (10 μg/ml; R&D Systems Inc.), andanti-IL-12 (10 μg/ml; R&D Systems Inc.). This experiment showed that, infact, antibody 2C8.vX bound to human Th17 cells as well as human Th1cells, but not to resting cells. See FIG. 12C-E.

EXAMPLE 13 Anti-LTα Antibodies Bind to Human Cells: T, B and NK A

A FACS assay was used to determine binding of anti-LTα antibodies to theLTα on the surface of primary human cells. Human PBMC were isolated andactivated with anti-CD3 and anti-CD28 (10 μg/ml and 5 μg/ml,respectively; BD PharMingen) for two days for activated CD4 and CD8cells; or anti-IgM (10 μg/ml, R&D Systems Inc.) and IL-4 (20 ng/ml; R&DSystems Inc.) for two days for B cells. NK CD56+ cells were isolated bynegative selection (Miltenyi Biotec) and incubated with IL-15 (20 ng/ml;R&D Systems Inc.) for 15 hours.

For FACS analysis, cells were incubated with chimeric anti-LTα antibody3F12 (1 μg/ml), human LTβR.huIgG1 (1 μg/ml), or huIgG1 isotype control(trastuzumab, 1 μg/ml), all directly conjugated to ALEXA FLUOR® 647fluorophore (Invitrogen Corp.), for 20 minutes in PBS with 2% FBS. Cellswere washed, aspirated, and re-suspended in PBS with 2% FBS. Cells wereanalyzed on a FACSCALIBUR™ (dual laser) using CELLQUEST™ software, andresults analyzed on a FLOWJO™ analyzer (Treestar).

FIGS. 13A-13E show that the antibodies bound to the cells, versus theisotype control. FIG. 13A represents the unactivated cells, FIG. 13Bshows the anti-CD3 and anti-CD28-activated CD4 T cells, FIG. 13C showsthe CD8 T cells, FIG. 13D shows the IL15-activated CD56 NK cells, andFIG. 13E shows the IgM and CD40L-activated CD19 B cells.

EXAMPLE 14 Anti-LTα Monoclonal Antibodies Functionally Block LTα3

1. Blocking of Human and Murine LTα3

Microtiter wells were coated with 0.7 μg/ml human TNFRII.huIgG1(ENBREL®) in 50 mM carbonate buffer solution (100 μl/well) overnight.The unabsorbed solution was aspirated from the wells. Biotinylated LTα3(murine or human) was captured and detected with streptavidin-horseradish peroxidase (HRP). For neutralization of human LTα3, increasingdoses of chimeric anti-LTα antibodies 2C8 or 3F12 or control isotype(trastuzumab) were preincubated with the LTα3 at indicatedconcentrations for one hour before adding to the coated microplate. Thesame procedure was carried out for neutralization of murine LTα3 usingthe hamster-mouse chimeric anti-LTα antibody S5H3 or anti-LT.Fc mutantor control isotype (trastuzumab). Each well was washed with PBS/0.05%TWEEN™ 20 surfactant, and bound HRP was measured with a solution oftetramethylbenzidine (TMB)/H₂O₂. After 15 minutes, the reaction wasquenched by the addition of 100 μl of 1M phosphoric acid to each well.The absorbance at 450 nm was read with a reference wavelength of 650 nm.

FIG. 14A shows that anti-LTα antibodies 2C8 and 3F12 functionallyblocked human LTα3 versus the isotype control. FIG. 14B shows thatanti-LTα antibody S5H3 and the anti-LT.Fc mutant functionally blockedmurine LTα3 versus the isotype control.

2. Blocking of LTα3-Induced IL-8 in A549 Cells

Epithelial cell lines (A549) were cultured in the presence of LTα3 for24 hours. Neutralization of the effect of LTα3 with chimeric anti-LTαantibodies 2C8 and 3F12 was determined by serial dilutions of theanti-LTα antibodies (10-0 μg/ml) added to the appropriate wells.Supernatant was used to detect IL-8 in a standard ELISA.

FIG. 14C shows that chimeric anti-LTα antibodies 2C8 and 3F12 blockedLTα3-induced IL-8 in A549 cells versus the isotype control (trastuzumabIgG1).

3. Blocking of LTα3-Induced ICAM Expression on HUVEC

HUVEC cells were cultured in the presence of LTα3 for 24 hours.Neutralization of the effect of LTα3 with chimeric anti-LTα antibodies2C8 and 3F12 was determined by serial dilutions of the anti-LTαantibodies (10-0 μg/ml) added to the appropriate wells. Cell-surfaceexpression of ICAM-1 was determined by FACS, shown as mean fluorescentintensity. TNFRII.Fc was used as a control.

FIG. 14D shows that anti-LTα antibodies 2C8 and 3F12, as well as theTNFRII.Fc mutant, blocked LTα3-induced ICAM expression on HUVEC versusthe isotype control (trastuzumab IgG1). The effect on unstimulated cellsis also shown.

4. Blocking of LTα3-Induced NFkB Activation

To determine if anti-LTα antibodies block signaling of LTα3 through theTNF receptors, 293 cell-lines were cultured in DMEM:F12 50:50 (+10% FBS,glutamine, P/S) to 75% confluency. Cells were transfected with astandard NFkB-RE-luciferase reporter construct using standardtransfection techniques (Fugene). After 24 hours, the cells were washed,and then stimulated with LTα3 (100 ng/ml) or with serial dilutions ofthe chimeric anti-LTα antibody 2C8 (10-0 μg/ml) that were preincubatedwith LTα3 for 30 minutes. After six hours, the cells were washed withPBS, and luciferase activity was measured as follows: LYSIS BUFFER™(Promega, 125 μl/well) was added for 15 minutes. Lysates were collectedand transferred at 20 μl/well to a 96-well assay plate (high-binding,white polystyrene, COSTAR™) in triplicates and read in a LMAXII™ platereader (Molecular Devices) after injection of LUCIFERASE ASSAY REAGENT™and STOP AND GLO BUFFERS™ (Promega). The isotype control used wastrastuzumab IgG1.

FIG. 14E shows that the anti-LTα antibody 2C8 blocked LTα3-induced NFkBactivation, versus the isotype control. The effect on unstimulated cellsis also shown.

EXAMPLE 15 Anti-LTα Antibodies Functionally Block LTα1β2

1. Blocking of LTα-Induced NFkB Activation

To determine if anti-LTα antibody blocks signaling of LTα1β2 through theLTβR, 293 cell lines were cultured in DMEM:F12 50:50 (+10% FBS,glutamine, P/S) to 75% confluency. Cells were transfected with standardNFkB-RE-luciferase reporter construct using standard transfectiontechniques (Fugene). After 24 hours, the cells were washed, and thenstimulated with LTα1β2 (R&D Systems Inc.; 100 ng/ml) or with serialdilutions of the chimeric anti-LTα antibody 2C8 (10-0 μg/ml) that werepreincubated with LTαβ for 30 minutes. After six hours, the cells werewashed with PBS, and luciferase activity was measured as follows: LYSISBUFFER™ (Promega, 125 μl/well) was added for 15 minutes. Lysates werecollected and transferred at 20 μl/well to a 96-well assay plate(high-binding, white polystyrene, COSTAR™) in triplicates and read in aLMAXII™ plate reader (Molecular Devices) after injection of LUCIFERASEASSAY REAGENT™ and STOP AND GLO BUFFERS™ (Promega). The isotype controlused was trastuzumab IgG1.

FIG. 15A shows that anti-LTα antibody 2C8 functionally blockedLTα1β2-induced NFkB activation, versus the isotype control. The responseof unstimulated cells is also shown.

2. Blocking of LTα3-, LTα2β1-, and LTα1β2-Induced Cytotoxicity

Test samples were assayed for ability to neutralize the cytolyticactivity of LTα3, LTα2β1, and LTα1β2 (R&D Systems Inc.) in a murine L929cytotoxic assay. L929 cells were cultured in microtiter plates in thepresence of one of the three LTα reagents (at the doses indicated inFIGS. 15B-D) and the DNA inhibitor actinomycin-D. Neutralizing chimericanti-LTα antibody 2C8 was added to the cultures at 10 μg/ml. Cell lysiswas determined by standard ALAMARBLUE™ stain of viable cells andrepresented as relative fluorescence units (RFU).

FIGS. 15B, 15C, and 15D show that chimeric anti-LTα antibody 2C8blocked, respectively, LTα3-, LTα2β1-, and LTα1β2-induced cytotoxicity.

3. Blocking of LTα 1β2-Induced ICAM Expression on HUVEC

HUVEC cells were cultured in the presence of LTα 1β2 (R&D Systems Inc.)for 24 hours. Neutralization of the effect of LTα1β2 with anti-LTαantibodies was determined by serial dilutions of chimeric anti-LTαantibodies 2C8 and 3F12 (10-0 μg/ml) added to the appropriate wells.Cell-surface expression of ICAM-1 was determined by FACS.

FIG. 15E shows that chimeric anti-LTα antibodies 2C8 and 3F12 blockedLTα1β2-induced ICAM expression on HUVEC, versus the isotype control(trastuzumab IgG1). The effect on unstimulated cells is also shown.

EXAMPLE 16 Anti-LTα Monoclonal Antibody can Kill LTαβ-Expressing Cells

An ADCC assay was performed in a microtiter plate in duplicate asfollows. NK cells were isolated from 100 ml of normal human donor wholeblood using negative selection (ROSETTESEP™, #15065, StemCellTechnologies). The assay diluent was RPMI™ 1640 and 0.25 mg/mL BSA.Chimeric antibody 2C8 and the Fc mutant thereof, in a serial dilutionstarting at 100 nM in 50 μl, were incubated with 50 μl of stable 293cells expressing human LTαβ (20,000) (see Example 11 for details) for 30minutes at room temperature. 50 μL of NK cells (120,000) were added andincubated for an additional four hours at 37° C. Plates were centrifugedat 1500 rpm for 10 minutes, and 100 μl of supernatant was transferred toa 96-flat-bottom microwell plate. The level of cell lysis was determinedby measuring the amount of lactate dehydrogenase (LDH kit, #1-644-793,Roche) released from lysed cells. 100 μl of LDH kit reaction mixture wasadded to 100 μl of supernatant and incubated for up to 30 minutes.Plates were read at 490 nm. Controls included target:effector cells inthe absence of antibody (for antibody-independent lysis), target cellsalone with 1% TRITON X-100™ surfactant (for total lysis), and antibodyADCC negative and positive controls.

FIG. 16 shows that anti-LTα antibody 2C8 lysed a 293-humanLTαβ-expressing cell line in the ADCC assay, but the corresponding Fcmutant monoclonal antibody did not.

EXAMPLE 17 Anti-LTα Monoclonal Antibody Blocks Cytokine and ChemokineSecretion in 3T3 and HUVEC Cells

HUVEC and 3T3 cells were cultured in the presence of human LTα3 (100ng/ml; R&D Systems Inc.) or human LTα1β2 (200 ng/ml; R&D Systems Inc.)for 24 hours using human or murine reagents, respectively.Neutralization of the effect of LTα trimers with chimeric 2C8 or S5H3anti-LTα antibodies (chimeric 2C8 with HUVEC, and hamster-mouse chimericS5H3 with murine 3T3 cells) was determined using a static dose of 10-0μg/ml. Supernatants were assayed for human RANTES, IL-6, IP-10, and IL-8for the HUVEC evaluation; or murine RANTES, IL-6, IP-10, and KC for the3T3 evaluation, using standard LINCOPLEX™ kits and read on a LUMINEX™plate reader.

FIG. 17A-H show that the anti-LTα antibody, versus an isotype controland as compared to unstimulated cells, blocked cytokine and chemokinesecretion in HUVEC cells, with FIGS. 17A-17D, respectively, showingKC/IL-8, RANTES, IP10, and IL-6 with LTα1β2, and FIGS. 17E-17H,respectively, showing KC/IL-8, RANTES, IP10, and IL-6 with LTα3.

FIG. 17I-P show that the anti-LTα antibody, versus an isotype controland as compared to unstimulated cells, blocks cytokine and chemokinesecretion in 3T3 cells, with FIGS. 17I-17L, respectively, showing KC,RANTES, IP10, and IL-6 with LTα1β2, and FIGS. 17M-17P, respectively,showing KC, RANTES, IP10, and IL-6 with LTα3.

In summary, the preferred antibodies herein against LTα:

-   -   1) Block LTα3    -   2) Deplete LTα-positive cells by binding LTα as target, which is        complexed with LTβ on the cell surface. The data that depletion        is important are the in vivo data comparing wild-type with the        Fc (DANA) mutant—for mouse: CIA, AIA (FIGS. 4 and 5); for human:        human SCID mice with 2C8 (FIG. 9) and ADCC assay (FIG. 16)    -   3) Block LTαβ function    -   4) Target any cell expressing LTα, including T-, B-, and        possibly or likely any Th1- or Th17- or Th2-driven disease (and        NK cells), without being limited to any one theory.

Diseases expected to benefit from treatment with the antibodies hereininclude RA, IBD, psoriasis, MS, Sjögren's syndrome, Hashimoto'sthyroiditis, Graves' disease, myasthenia gravis, and lupus, especially,RA, MS, lupus (e.g., SLE and LN), and IBD, including Crohn's disease andUC.

EXAMPLE 18 Affinity-Matured Anti-LTα Antibody 2C8.vX

Affinity maturation of the antibody 2C8 noted in Example 3 led to thehumanized antibody 2C8.vX. The light-chain and heavy-chain variableregions and CDRs of this antibody are shown in FIGS. 18A and 18B,respectively, along with the CDRs.

The following is the DNA sequence that encodes the full-length heavychain of anti-LTα 2C8.vX:

(SEQ ID NO: 104) GAAGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCC TGTGCAGCTTCTGGCTACACATTCACCAGCTATGTGATCCATTGGGTCCGTCAGGCCCCGGGTAAGGGCCTCGAGTGGGTTGGTTATAACAATCCTTATAACGCCGGCACCAACTATAACGAGAAGTTCAAGGGGCGCTTCACTATCAGTTCTGACAAGTCGAAAAACACAGCATACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTCGACCCACAATGCTCCCATGGTTCGCCTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCAGCCTCC ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAG CGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT CAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCT CCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCTCCGGGTAAA

The following is the DNA sequence that encodes the full-length lightchain of anti-LTα 2C8.vX:

(SEQ ID NO: 105) GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGCTGTGTCTTCCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCTGCATCCCACCGTTACACTGGAGTCCCTTCTCGCTTCTCTGGATCCGGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGGAATCTTATTCTACTCCTTGGACGTTCGGAC AGGGTACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG AGAGTGT

The following is the full-length heavy-chain amino acid sequence for2C8.vX:

(SEQ ID NO: 106) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYVIHWVRQAPGKGLEWVGYNNPYNAGTNY NEKFKGRFTISSDKSKNTAYLQMNSLRAEDTAVYYCSRPTMLPWFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The following is the full-length light-chain amino acid sequence for2C8.vX:

(SEQ ID NO: 107) DIQMTQSPSSLSASVGDRVTITCRASQAVSSAVAWYQQKPGKAPKLLIYSASHRYTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQESYSTPWTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Epitope mapping indicates that this molecule has a partial overlap withthe receptor binding site. Activated rhesus T and B cells expresssurface LTαβ. The antibody 2C8.vX bound (crossreacted with) rhesussurface LTαβ (as determined by FACS) and rhesus soluble LTα3 (asdetermined by FORTEBIO™ octet).

FIGS. 19A and 19B show, respectively, ELISAs of LTα3 and LTα1β2 bindingof 2C8.vX antibodies in comparison to isotype control/TNFRII.Fc. TheseELISAs were performed in the same way as described in Example 10 aboveusing human LTα3 and in Example 11 using LTα1β2. The EC₅₀ values ofthese same antibodies to LTβ3 and LTα1β2 are 1.03 nM for TNFRII.Fc and0.05 nM for 2C8.vX to LTα3, and no binding of TNFRII.Fc and 0.34 nM for2C8.vX to LTα1β2. The results show that 2C8.vX bound much better thanTNFRII.Fc in both cases.

FIGS. 20A and 20B show, respectively, blocking of human LTα3 and LTα1β2by 2C8vX antibodies in comparison to isotype control/TNFRII.Fc using theassay for testing blocking of human LT-induced cytotoxicity described inExample 15 above. The IC₅₀ values of these same antibodies to LTα3 andLTα1β2 are 0.83 nM for TNFRII.Fc and 0.29 nM for 2C8.vX to LTα3, with noisotype blocking, and 0.31 nM for 2C8.vX to LTα1β2, with no isotypeblocking. The results show that 2C8.vX blocked LTα3 better thanTNFRII.Fc.

FIG. 21 relates to a functional assay showing inhibition of ICAM1upregulation by LTα3 on HUVEC cells, using the protocol described inExample 14 above. FIG. 21 shows a comparison of how various moleculesblocked LTα3 in this functional assay. Specifically, isotype control,2C8.vX, and TNFRII.Fc were compared. The results show that bothantibodies were able to block LTα3 in this assay.

FIG. 22 relates to a functional assay showing blocking of LTα1β2 using293-LTβR cells, using the protocol described in Example 15 above. FIG.22 shows a comparison of how various molecules blocked LTβ1β2 in thisfunctional assay. Specifically, isotype control and 2C8.vX were comparedalong with unstimulated cells. The results show that 2C8.vX was able toblock LTα1β2 in this assay, with an IC₅₀ of about 5 nM.

FIG. 23 shows ADCC activity of 2C8.vX using the protocol set forth inExample 16 involving 293-LTα1β2 cells. 2C8.vX was compared with the2C8.vX DANA mutant and isotype control. The results show that 2C8.vX hadADCC activity, whereas the DANA mutant did not.

FIG. 24 shows GVHD survival results with 2C8.vX and CTLA4-Fc, ascompared to controls (Fc mutant and isotype control) in a human SCIDmodel, using the same protocol as described above in Example 9,terminating in three to four weeks. The results indicate that CTLA4-Fcand 2C8.vX prolonged survival in this model, and thus were effective atdepleting LTα cells, but not the controls 2C8.vX-DANA mutant and humanIgG1 isotype control antibody.

FIG. 25A-D show that the 2C8.vX antibody depleted LTα1β2-expressing Tand B cells in the human SCID GVHD model. In this protocol, SCID micewere reconstituted with PBMCs purified from a LEUKOPACK™ (available fromblood banks such as Interstate Blood Bank, Memphis, Tenn.) of a normaldonor by FICOLL™ polysaccharide gradient. All mice (n=10/group) weresub-lethally irradiated with 350 rads using a Cesium 137 source. Twohours after irradiation, mice were injected with 50 million humanPBMCs/mouse in 200 μl PBS intrasplenically. One day after cellinjection, mice were treated i.p. either with 300 μg of human IgG1isotype control antibody, 2C8.vX, or 2C8.vX DANA mutant in 100 μl salinefor one day. On Day 2 after irradiation, the splenocytes of the micewere labeled with 5-(and 6-)carboxy fluorescein diacetate succinimidylester (CFSE), and the absolute cell number (total CFSE positive cells)was measured and the percentage of cells in CFSE peaks was measuredafter electronically gating on CD4⁺, CD8⁺, and CD19⁺ cells. For thisthree-color staining, the following monoclonal antibodies were used:PE-labeled anti-CD4, PE-labeled anti-CD8, and PE-labeled CD19. Stainedcells were analyzed on a FACSCAN™ or FACSCALIBUR™ instrument.

The results in FIG. 25A show total CFSE-positive human cells at day 2for isotype control, 2C8.vX, and 2C8.vX.DANA mutant, and indicate thatdepletion was best with the vX antibody. FIG. 25B-D show the results ofgating using the CD4, CD8, and CD19 antibodies, respectively, indicatingthat the vX antibody was best at depleting LTαβ-positive T and B cellsas compared to isotype control and the DANA mutant.

In summary, the antibody 2C8.vX depletes LT-expressing cells in an invitro ADCC assay and in vivo in two human SCID GVHD models. It alsoblocks LTα3 and LTα1β2 in functional cell assays using HUVEC cells and293-LTβR cells, respectively. Further, it cross-reacts with non-humanprimate (NHP) lymphotoxin as determined by activated rhesus primarycells and the LTα3 rhesus protein.

EXAMPLE 19 Affinity Matured Anti-LTα Antibody 3F12.v14

Affinity maturation of the antibody 3F12 noted above led to thehumanized antibody 3F12.v14. The light-chain and heavy-chain variableregions and CDRs of this antibody are shown in FIGS. 26A and 26B,respectively, along with the CDRs.

When tested for binding and blocking LTα3 and LTα using the assays notedabove, 3F12.v14 bound and blocked these molecules as well as didantibody 2c8.vX.

EXAMPLE 20 Afucosylated 2C8.vX

The antibodies herein may be altered so that they lack fucose byculturing them using a cell line or technology disclosed in US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; US 2006/0063254; US2006/0064781; US 2006/0078990; US 2006/0078991; Okazaki et al. J. Mol.Biol. 336:1239-1249 (2004); or Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004). Examples of cell lines producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al., Arch. Biochem. Biophys., 249:533-545 (1986); US2003/0157108 A1 (Presta) and WO 2004/056312 A1 (Adams et al., especiallyat Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knock-out CHO cells(Yamane-Ohnuki et al. supra).

In fact, the antibody 2C8.vX was afucosylated to produce an antibodycalled anti-LTa 2C8.vX AF. This molecule was generated using a stableCHO cell line having a FUT8 knock-out gene as described by the method inYamane-Ohnuki et al., supra It is 100% afucosylated.

The biophysical properties of 2C8.vX (including blocking and bindingdata) were not substantially changed upon afucosylation thereof, exceptfor the ADCC. The ADCC properties of the wild-type and afucosylatedmolecules were determined by carrying out the experiments using theprotocol set forth in Example 16 involving 293-LTα1β2 cells. The resultsare shown in FIG. 27. The figure shows that the afucosylated antibodyanti-LTa 2C8.vX AF has about a ten-fold increase over the wild-type (WT)antibody (produced in a normal CHO cell line as described above).

EXAMPLE 21 Underfucosylated 2C8.vX

As an alternative way to achieve high yields of non-fucosylatedantibodies in mammalian cells, an RNAi approach may be employed to knockdown the expression of the FUT8 gene. A plasmid may be used to produceshort hairpin siRNA consisting of 19 nt (nucleotide) sense siRNAsequence specific to the gene of FUT8, linked to its reversecomplementary antisense siRNA sequence by a short spacer (9nt hairpinloop), followed by 5-6 U's at the 3′ end.

Four different RNAi probes are designed to target the different regionsbased on the available CHO FUT8 DNA sequence.

For testing the efficacy of these RNAi probes, a FLAG-tagged FUT8 fusionprotein is constructed using the available CHO FUT8 DNA sequence(Genbank accession no. P_AAC63891). RT-PCR is performed with FUT8primers and the resulting PCR fragment fused with 5′ FLAG tag sequence.The tagged FUT8 fragment is cloned into an expression vector. The RNAiprobe plasmid and flag-tagged FUT8 plasmid are cotransfected into CHOcells. Cell lysate is extracted 24 hours after transfection and the FUT8fusion protein level analyzed using anti-flag M2 antibody byimmuno-blotting. In the presence of RNAi probes, the fusion proteinexpression is expected to be significantly inhibited in most of thecases.

The two probes showing the best inhibitory effect are transfected into aCHO cell line expressing an antibody as described herein, such as2C8.vX. The expressed anti-LT-alpha antibody is purified by a protein Acolumn and submitted for Matrix-Assisted Laser Desorption/IonizationTime-of-flight (MALDI-TOF) mass spectral analysis of asparagine-linkedoligosaccharides (including fucose content) and FcγR binding assay asdescribed below.

Methods for analyzing the oligosaccharides by MALDI-TOF are conductedgenerally as follows: N-linked oligosaccharides are released fromrecombinant glycoproteins using the peptide-N-glycosidase-F (PNGase F)procedure of Papac et al., Glycobiology 8, 445-454 (1998). Briefly, thewells of a 96-well polyvinylidene difluoride (PVDF)-lined microtitreplate (Millipore, Bedford, Mass.) are conditioned with 100 μl methanolthat is drawn through the PVDF membranes by applying vacuum to theMILLIPORE™ multiscreen vacuum manifold. The conditioned PVDF membranesare washed with 3×250 μl water. Between all wash steps the wells aredrained completely by applying gentle vacuum to the manifold. Themembranes are washed with reduction and carboxymethylation buffer (RCM)consisting of 6 M guanidine hydrochloride, 360 mM TRIS buffer, 2 mMEDTA, pH 8.6. Glycoprotein samples (50 μg) are applied to individualwells, again drawn through the PVDF membranes by gentle vacuum and thewells washed with 2×50 μl of RCM buffer. The immobilized samples arereduced by adding 50 μl of a 0.1 M dithiothreitol (DTT) solution to eachwell and incubating the microtitre plate at 37° C. for 1 hr. DTT isremoved by vacuum and the wells are washed 4×250 μl water. Cysteineresidues are carboxylmethylated by the addition of 50 μl of a 0.1 Miodoacetic acid (IAA) solution that is freshly prepared in 1 M NaOH anddiluted to 0.1 M with RCM buffer.

Carboxymethylation is accomplished by incubation for 30 min in the darkat ambient temperature. Vacuum is applied to the plate to remove the IAAsolution and the wells are washed with 4×250 μl purified water. The PVDFmembranes are blocked by the addition of 100 μl of 1% PVP360™(polyvinylpyrrolidine 360,000 MW) (Sigma) solution and incubation for 30minutes at ambient temperature. The PVP-360™ solution is removed bygentle vacuum and the wells are washed 4×250 μl water. Peptide:N-Glycosidase F (PNGase F™) amidase (New England Biolabs, Beverly,Mass.), at 25 μl of a 25 Unit/ml solution in 10 mM TRIS acetate, pH 8.3,is added to each well and the digest proceeds for 3 hr at 37° C. Afterdigestion, the samples are transferred to 500 μl EPPENDORF™ tubes and2.5 μl of a 1.5 M acetic acid solution is added to each sample. Theacidified samples are incubated for two hrs at ambient temperature toconvert the oligosaccharides from the glycosylamine to the hydroxylform. Prior to MALDI-TOF mass spectral analysis, the releasedoligosaccharides are desalted using a 0.7-ml bed of cation-exchangeresin (AG50W-X8 resin in the hydrogen form) (Bio-Rad, Hercules, Calif.)slurry packed into compact reaction tubes (US Biochemical, Cleveland,Ohio).

For MALDI-TOF mass-spectral analysis of the samples in the positivemode, the desalted oligosaccharides (0.5 μl aliquots) are applied to thestainless target with 0.5 μl of the 2,5 dihydroxybenzoic acid matrix(sDHB) that is prepared by dissolving 2 mg 2,5 dihydroxybenzoic acidwith 0.1 mg of 5-methoxyslicylic acid in 1 ml of 1 mM NaCl in 25%aqueous ethanol. The sample/matrix mixture is vacuum dried and thenallowed to absorb atmospheric moisture prior to analysis. Releasedoligosaccharides are analyzed by MALDI-TOF on a PERSEPTIVE BIOSYSTEMSVOYAGER-ELITE™ mass spectrometer. The mass spectrometer is operated inthe positive mode at 20 kV with the linear configuration and utilizingdelayed extraction. Data are acquired using a laser power ofapproximately 1100 and in the data summation mode (240 scans) to improvethe signal to noise ratio. The instrument is calibrated with a mixtureof standard oligosaccharides and the data are smoothed using a 19-pointSavitsky-Golay algorithm before the masses are assigned. Integration ofthe mass-spectral data is achieved using the CAESAR7.2™ data analysissoftware package (SciBridge Software).

Binding of control and test materials to the human Fcγ receptors can beassessed using modified versions of procedures originally described byShields et al., J Biol Chem, 276:6591-604 (2001).

For confirmation that the RNAi-transfected cells do have less FUT8 RNAexpression, a Northern blot can be performed using RNA samples extractedfrom the transfected cells 24 hours after transfection. Total RNA fromcells containing a control plasmid (random mouse DNA sequence, nohomology to any known mouse proteins) and two RNAi plasmids can bepurified and hybridized with a 300-bp probe. The knock down ofendogenous α 1,6-fucosyltransferase RNA can be further confirmed byquantitative PCR.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC):

ATCC Material Dep. No. Deposit Date hybridoma murine LymphotoxinPTA-7538 Apr. 19, 2006 alpha2 beta1 s5H3.2.2

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the example presented herein.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

1. A DNA encoding an isolated anti-lymphotoxin-α(LTα) antibodycomprising a light chain comprising the amino acid sequence of SEQ IDNO:107 and a heavy chain comprising the amino acid sequence of SEQ IDNO:106.
 2. An isolated nucleic acid encoding the heavy chain of theantibody of claim
 1. 3. An expression vector comprising the nucleic acidof claim
 2. 4. An isolated host cell comprising the nucleic acid ofclaim
 2. 5. A method of producing a heavy chain of an antibodycomprising culturing the host cell of claim 4 under conditions toproduce the heavy chain of the antibody and recovering the heavy chainof the antibody.
 6. An isolated nucleic acid encoding the light chain ofthe antibody of claim
 1. 7. An expression vector comprising the nucleicacid of claim
 6. 8. An isolated host cell comprising the nucleic acid ofclaim
 6. 9. A method of producing a light chain of an antibodycomprising culturing the host cell of claim 8 under conditions toproduce the light chain of the antibody and recovering the light chainof the antibody.
 10. A method of producing an antibody comprising: a.culturing a host cell comprising nucleic acid encoding ananti-lymphotoxin-αantibody having the light chain comprising SEQ IDNO:107 and a heavy chain comprising SEQ ID NO:106 under conditions toproduce the antibody; and b. recovering the antibody.
 11. The method ofclaim 10 wherein the light and heavy chains are encoded on separatevectors.
 12. The method of claim 10 wherein the light and heavy chainsare encoded on the same vector.
 13. An antibody produced by the methodof claim 10.